Stable hydrazones linked to a peptide moiety as reagents for the preparation of radiopharmaceuticals

ABSTRACT

This invention provides novel reagents for the preparation of radiopharmaceuticals useful as imaging agents for the diagnosis of cardiovascular disorders, infection, inflammation and cancer, diagnostic kits comprising said reagents and intermediate compounds useful for the preparation of said reagents. The reagents are comprised of stable hydrazone modified biologically active molecules that react with gamma emitting radioisotopes to form radiopharmaceuticals that selectively localize at sites of disease and thus allow an image to be obtained of the loci using gamma scintigraphy.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of our applicationU.S. Ser. No. 08/218,861 filed Mar. 28, 1994, pending which is acontinuation-in-part of U.S. Ser. No. 08/040,336 filed Mar. 30, 1993,now abandoned, the disclosures of which are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to novel reagents for the preparation ofradiopharmaceuticals useful as imaging agents for the diagnosis ofcardiovascular disorders, infection, inflammation and cancer, todiagnostic kits comprising said reagents and to intermediate compoundsuseful for the preparation of said reagents. The reagents are comprisedof stable hydrazone modified biologically active molecules that reactwith gamma emitting radioisotopes to form radiopharmaceuticals thatselectively localize at sites of disease and thus allow an image to beobtained of the loci using gamma scintigraphy.

BACKGROUND OF THE INVENTION

There is a current need for new methods for the non-invasive diagnosisof a variety of diseases such as thromboembolic disease,atherosclerosis, infection and cancer. Radiopharmaceuticals comprised ofgamma-ray emitting radionuclide labeled biologically active moleculescan fulfill the need. The biologically active molecules serve tolocalize the radionuclides at the sites of disease and thus allow thesites to be visualized by gamma scintigraphy. The molecules can beeither proteins, antibodies, antibody fragments, peptides orpolypeptides, or peptidomimetics. The molecules interact with a receptoror binding site expressed at the sites of the disease or with a receptoror binding site on an endogenous blood component, such as platelets andleukocytes, that accumulate at the sites. This interaction results inselective localization of a percentage of the injectedradiopharmaceutical while the remainder is cleared either through therenal or hepatobiliary systems. The localized radiopharmaceutical isthen imaged externally using gamma scintigraphy. The relative rates oflocalization, clearance and radionuclidic decay determine the ease ofvisualization, often expressed as the target-to-background ratio.Frequently, only certain portions of the biologically active moleculesbind to the receptors; these portions are termed the recognitionsequences or units.

A number of radiopharmaceuticals comprised of radionuclide labeledproteins, antibodies or antibody fragments are under development,however, to date only one has been approved by the Food and DrugAdministration. This sparse record results from a combination of factorsthat make developing these radiopharmaceuticals difficult, includingproblems with manufacturing and quality control, non-optimalsequestration and clearance rates, and the occurence of antigenic orallergic responses to the radiopharmaceuticals. These problems aremainly due to the macromolecular nature of the proteins, antibodies andantibody fragments. Their high molecular weight makes direct chemicalsynthesis impractical, therefore they must be synthesized by recombinantor cloning techniques that typically give low yields and requireextensive isolation and purification procedures. Their molecular weightcan slow their rates of localization and preclude their clearance by anactive elimination mechanism via the kidneys or liver, resulting inprolonged retention in the circulation which causes a high backgroundlevel during imaging. Also, the body's immune system tends to recognizemore efficiently larger exogenous species.

The use of lower molecular weight peptides, polypeptides orpeptidomimetics as the biologically active molecules obviates a numberof these problems. These molecules can be synthesized directly usingclassical solution chemistry or by an automated peptide synthesizer.They can be formed in higher yields and require less complicatedpurification procedures. They tend to clear more rapidly from thecirculation by an active elimination pathway resulting in a lowerbackground in the images. They are also usually not immunogenic. Thefirst radionuclide labeled polypeptide radiopharmaceutical has beenrecently approved by the Food and Drug Administration.

There are two general methods for labeling biologically active moleculeswith radionuclides for use as radiopharmaceuticals termed direct andindirect labeling. Direct labeling involves attaching the radionuclideto atoms on the biologically active molecule; while the indirect methodinvolves attaching the radionuclide via a chelator. The chelator caneither be attached to the biologically active molecule prior to reactionwith the radionuclide or the radionuclide labeled chelator moiety can beattached to the biologically active molecule. Several recent reviewsdescribe these labeling methods and are incorporated herein byreference: S. Jurisson et. al., Chem. Rev., 1993, 93, 1137; A.Verbruggen, Eur. J. Nuc. Med., 1990, 17, 346; and M. Derwanjee, Semin.Nuc. Med., 1990, 20, 5.

The use of hydrazines and hydrazides as chelators to modify proteins forlabeling with radionuclides has been recently disclosed in Schwartz et.al., U.S. Pat. No. 5,206,370. The protein is modified by reaction withbifunctional aromatic hydrazines or hydrazides having a protein reactivesubstituent. For labeling with technetium-99m, the hydrazino-modifiedprotein is reacted with a reduced technetium species, formed by reactingpertechnetate with a reducing agent in the presence of a chelatingdioxygen ligand. The technetium becomes bound to the protein throughwhat are believed to be hydrazido or diazenido linkages with thecoordination sphere completed by the ancillary dioxygen ligands.Examples of ancillary dioxygen ligands include glucoheptonate,gluconate, 2-hydroxyisobutyrate, and lactate.

In one embodiment of the invention described in Schwartz et. al., thebifunctional aromatic hydrazine or hydrazide is protected as a loweralkyl hydrazone. This was done to prevent cross-reaction between thehydrazine or hydrazide and the protein reactive substituent because inthe absence of the protecting group the bifunctional compound reactswith a protein to form a hydrazone modified protein. The free hydrazineor hydrazide group on the protein is then formed by dialysis into anacidic (pH 5.6) buffer and mixed with a suitable metal species, such asa reduced technetium species, in acidic media to yield a labeledprotein.

Although the lower alkyl hydrazone protecting group prevents thecross-reaction between the hydrazine or hydrazide and the proteinreactive substituent, it can be displaced by other aldehydes and ketonesto form different hydrazones. This is a serious and significantdisadvantage. The presence of other aldehydes and ketones in smallquantities is unavoidable in a commercial pharmaceutical manufacturingsetting, because they are extracted from various plastic and rubbermaterials and are also used in common disinfectants. Small quantities offormaldehyde are particularly ubiquitous. Therefore, reagents comprisedof lower alkyl hydrazone protected biologically active molecules candegrade into a number of different hydrazone containing species,depending on the number and quantities of other aldehydes and ketones towhich they are exposed during processing or manufacturing or storageafter manufacture. This presents a significant problem in maintainingthe purity of the reagents, and thus renders lower alkyl protectedreagents unattractive as commercial candidates.

The present invention provides novel reagents for the preparation ofradiopharmaceuticals comprised of stable hydrazone modified biologicallyactive molecules. The stable hydrazones do not react appreciably withother aldehydes and ketones, maintaining the purity of the reagentsduring manufacturing. Surprisingly, these stable hydrazone reagents arestill reactive enough to be labeled with radionuclides such astechnetium-99m.

SUMMARY OF THE INVENTION

This invention relates to novel reagents for the preparation ofradiopharmaceuticals useful as imaging agents for the diagnosis ofcardiovascular disorders, infection, inflammation and cancer. Thereagents are comprised of stable hydrazone modified biologically activemolecules that react with gamma emitting radioisotopes to formradiopharmaceuticals that selectively localize at sites of disease andthus allow an image to be obtained of the loci using gamma scintigraphy.The stable hydrazone serves as a protecting group for the chelator orbonding unit of the reagents preventing decomposition or degradationduring the manufacturing process. This invention also providesdiagnostic kits comprising such reagents. This invention also providesnovel intermediate compounds useful for the preparation of saidreagents.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1. A comparison of the stability of the reagent described inExample 1 to 10 equivalents of formaldehyde with that of the lower alkylhydrazone compound,cyclo-(D-Val-NMeArg-Gly-Asp-Mamb(hydrazino-nicotinyl-5-Aca))propionaldehyde hydrazone.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides novel reagents for the preparation ofradiopharmaceuticals useful as imaging agents for the diagnosis ofcardiovascular disorders, infection, inflammation and cancer, diagnostickits comprising said reagents and intermediate compounds useful for thepreparation of said reagents. The reagents are comprised of stablehydrazone modified biologically active molecules that react with gammaemitting radioisotopes to form radiopharmaceuticals that selectivelylocalize at sites of disease and thus allow an image to be obtained ofthe loci using gamma scintigraphy.

1! One embodiment of this invention is a reagent for preparing aradiopharmaceutical comprising a biologically active group connected toa stable hydrazone group, optionally having a linking group between saidstable hydrazone and said biologically active group.

2! Another embodiment of this invention is the reagent of embodiment 1!having a linking group between said stable hydrazone and saidbiologically active group.

3! Another embodiment of this invention is the reagent of embodiment 2!having the formula:

    (Q)d'L.sub.n --H.sub.z,

and pharmaceutically acceptable salts thereof

wherein,

Q is a biologically active group;

d' is 1-20;

L_(n) is a linking group of formula:

    M.sup.1 -- Y.sup.1 (CR.sup.55 R.sup.56).sub.f (Z.sup.1).sub.f" Y.sup.2 !.sub.f' --M.sup.2,

wherein:

M¹ is -- (CH₂)_(g) Z¹ !_(g') --(CR⁵⁵ R⁵⁶)_(g") --;

M² is --(CR⁵⁵ R⁵⁶)_(g") -- Z¹ (CH₂)_(g) !_(g') --;

g is independently 0-10;

g' is independently 0-1;

g" is independently 0-10;

f is independently 0-10;

f' is independently 0-10;

f" is independently 0-1;

Y¹ and Y², are independently selected at each occurrence from: a bond,O, NR⁵⁶, C═O, C(═O)O, OC(═O)O, C(═O)NH--, C═NR⁵⁶, S, SO, SO₂, SO₃,NHC(═O), (NH)₂ C(═O), (NH)₂ C═S;

Z¹ is independently selected at each occurrence from a C₆ -C₁₄saturated, partially saturated, or aromatic carbocyclic ring system,substituted with 0-4 R⁵⁷ ; and a heterocyclic ring system, substitutedwith 0-4 R⁵⁷ ;

R⁵⁵ and R⁵⁶ are independently selected at each occurrence from:hydrogen; C₁ -C₁₀ alkyl substituted with 0-5 R⁵⁷ ; alkaryl wherein thearyl is substituted with 0-5 R⁵⁷ ;

R⁵⁷ is independently selected at each occurrence from the group:hydrogen, OH, NHR⁵⁸, C(═O)R⁵⁸, OC(═O)R⁵⁸, OC(═O)OR⁵⁸, C(═O)OR⁵⁸,C(═O)NR⁵⁸, C.tbd.N, SR⁵⁸, SOR⁵⁸, SO₂ R⁵⁸, NHC(═O)R⁵⁸, NHC(═O)NHR⁵⁸,NHC(═S)NHR⁵⁸ ; or, alternatively, when attached to an additionalmolecule Q, R⁵⁷ is independently selected at each occurrence from thegroup: O, NR⁵⁸, C═O, C(═O)O, OC(═O)O, C(═O)N--, C═NR⁵⁸, S, SO, SO₂, SO₃,NHC(═O), (NH)₂ C(═O), (NH)₂ C═S; and,

R⁵⁸ is independently selected at each occurrence from the group:hydrogen; C₁ -C₆ alkyl; benzyl, and phenyl;

Hz is a stable hydrazone of formula: ##STR1## wherein, R⁴⁰ isindependently selected at each occurrence from the group: a bond toL_(n), C₁ -C₁₀ alkyl substituted with 0-3 R⁵², aryl substituted with 0-3R⁵², cycloaklyl substituted with 0-3 R⁵², heterocycle substituted with0-3 R⁵², heterocycloalkyl substituted with 0-3 R⁵², aralkyl substitutedwith 0-3 R⁵² and alkaryl substituted with 0-3 R⁵² ;

R⁴ 1 is independently selected from the group: hydrogen, arylsubstituted with 0-3 R⁵², C₁ -C₁₀ alkyl substituted with 0-3 R⁵², and aheterocycle substituted with 0-3 R⁵² ;

R⁵² is independently selected at each occurrence from the group: a bondto L_(n), ═O, F, Cl, Br, I,--CF₃, --CN, --CO₂ R⁵³, --C(═O)R⁵³,--C(═O)N(R⁵³)₂, --CHO, --CH₂ OR⁵³, --OC (═O)R⁵³, --OC (═O)OR^(53a),--OR⁵³, --OC (═O)N(R⁵³)₂, --NR⁵³ C(═O)R⁵³, --NR⁵⁴ C(═O)OR^(53a), --NR⁵³C(═O)N(R⁵³)₂, --NR⁵⁴ SO₂ N(R⁵³)₂, --NR⁵⁴ SO₂ R^(53a), --SO₃ H, --SO₂R^(53a), --SR⁵³, --S(═O)R^(53a), --SO₂ N(R⁵³)₂, --N(R⁵³)₂,--NHC(═NH)NHR⁵³, --C(═NH)NHR⁵³, ═NOR⁵³, NO₂, --C(═O)NHOR⁵³,--C(═O)NHNR⁵³ R^(53a), --OCH₂ CO₂ H, 2-(1-morpholino)ethoxy;

R⁵³, R^(53a), and R⁵⁴ are each independently selected at each occurrencefrom the group: hydrogen, C₁ -C₆ alkyl, and a bond to L_(n) ;

R⁸⁰ and R⁸¹ are independently selected from the group: H; C₁ -C₁₀ alkyl;--CN; --CO₂ R⁸⁵ ; --C(═O)R⁸⁵ ; --C(═O)N(R⁸⁵)₂ ; C₂ -C₁₀ 1-alkenesubstituted with 0-3 R⁸⁴ ; C₂ -C₁₀ 1-alkyne substituted with 0-3 R⁸⁴ ;aryl substituted with 0-3 R⁸⁴ ; unsaturated heterocycle substituted with0-3 R⁸⁴ ; and unsaturated carbocycle substituted with 0-3 R⁸⁴ ; providedthat when one of R⁸⁰ and R⁸¹ is H or alkyl, then the other is not H oralkyl;

or, alternatively, R⁸⁰ and R⁸¹, may be taken together with the showndivalent carbon radical to form: ##STR2## wherein: R⁸² and R⁸³ may beindependently selected from the group: H; R⁸⁴ ; C₁ -C₁₀ alkylsubstituted with 0-3 R⁸⁴ ; C₂ -C₁₀ alkenyl substituted with 0-3 R⁸⁴ ; C₂-C₁₀ alkynyl substituted with 0-3 R⁸⁴ ; aryl substituted with 0-3 R⁸⁴ ;heterocycle substituted with 0-3 R⁸⁴ ; and carbocycle substituted with0-3 R⁸⁴ ;

or, alternatively, R⁸², R⁸³ may be taken together to form a fusedaromatic or heterocyclic ring;

a and b indicate the positions of optional double bonds and n is 0 or 1,

R⁸⁴ is independently selected at each occurrence from the group: ═O, F,Cl, Br, I, --CF₃, --CN, --CO₂ R⁸⁵, --C(═O)R⁸⁵, --C(═O)N(R⁸⁵)₂, --N(R⁸⁵)₃⁺ --CH₂ OR⁸⁵, --OC(═O)R⁸⁵, --OC(═O)OR^(85a), --OR⁸⁵, --OC(═O)N(R⁸⁵)₂,--NR⁸⁵ C(═O)R⁸⁵, --NR⁸⁶ C(═O)OR^(85a), --NR⁸⁵ C(═O)N(R⁸⁵)₂, --NR⁸⁶ SO₂N(R⁸⁵)₂, --NR⁸⁶ SO₂ R^(85a), --SO₃ H, --SO₂ R^(85a), --SR⁸⁵,--S(═O)R^(85a), --SO₂ N(R⁸⁵)₂, --N(R⁸⁵)₂, --NHC (═NH)NHR⁸⁵,--C(═NH)NHR⁸⁵, ═NOR⁸⁵, --C(═O)NHOR⁸⁵, --OCH₂ CO₂ H,2-(1-morpholino)ethoxy; and

R⁸⁵, R^(85a), and R⁸⁶ are each independently selected at each occurrencefrom the group: hydrogen, C₁ -C₆ alkyl.

4! Another embodiment of this invention is the reagent of embodiment 3!wherein:

Q is a biologically active molecule selected from the group: IIb/IIIareceptor antagonists, IIb/IIIa receptor ligands, fibrin bindingpeptides, leukocyte binding peptides, chemotactic peptides, somatostatinanalogs, and selectin binding peptides;

d' is 1 to 3;

L_(n) is:

--(CR⁵⁵ R⁵⁶)_(g") -- Y¹ (CR⁵⁵ R⁵⁶)_(f) Y² !_(f') --(CR⁵⁵ R⁵⁶)_(g") --,

wherein:

g" is 0-5;

f is 0-5;

f' is 1-5;

Y¹ and Y², are independently selected at each occurrence from: O, NR⁵⁶,C═O, C(═O)O, OC(═O)O, C(═O)NH--, C═NR⁵⁶, S, SO, SO₂, SO₃, NHC(═O), (NH)₂C(═O), (NH)₂ C═S;

R⁵⁵ and R⁵⁶ are independently selected at each occurrence from:hydrogen, C₁ -C₁₀ alkyl, and alkaryl;

H_(z) is a stable hydrazone of formula: ##STR3## wherein, R⁴⁰ isindependently selected at each occurrence from the group: arylsubstituted with 0-3 R⁵², and heterocycle substituted with 0-3 R⁵² ;

R⁴¹ is independently selected from the group: hydrogen, aryl substitutedwith 0-1 R⁵², C₁ -C₃ alkyl substituted with 0-1 R⁵², and a heterocyclesubstituted with 0-1 R⁵² ;

R⁵² is independently selected at each occurrence from the group: a bondto L_(n), --CO₂ R⁵³, --CH₂ OR⁵³, --SO₃ H, --SO₂ R^(53a), --N(R⁵³)₂,--NHC(═NH)NHR⁵³, and --OCH₂ CO₂ H;

R⁵³, R^(53a) are each independently selected at each occurrence from thegroup: hydrogen and C₁ -C₃ alkyl;

R⁸⁰ is independently selected at each occurrence from the group:

--CO₂ R⁸⁵ ;

C₂ -C₅ 1-alkene substituted with 0-3 R⁸⁴ ;

C₂ -C₅ 1-alkyne substituted with 0-3 R⁸⁴ ;

aryl substituted with 0-3 R⁸⁴ ;

unsaturated heterocycle substituted with 0-3 R⁸⁴ ;

R⁸¹ is independently selected at each occurrence from the group: H andC.sub. -C₅ alkyl;

or, alternatively, R⁸⁰ and R⁸¹, when taken together with the indicateddivalent carbon radical form ##STR4## wherein R⁸² and R⁸³ may beindependently selected from the group: H and R⁸⁴ ;

or, alternatively, R⁸², R⁸³ may be taken together to form a fusedaromatic or heterocyclic ring;

a and b indicate the positions of optional double bonds and n is 0 or 1,

R⁸⁴ is independently selected at each occurrence from the group: --CO₂R⁸⁵, --C(═O)N(R⁸⁵)₂, --CH₂ OR⁸⁵, --OC(═O)R⁸⁵ --OR⁸⁵, --SO₃ H, --N(R⁸⁵)₂,--OCH₂ CO₂ H;

R⁸⁵ is independently selected at each occurrence from the group:hydrogen, C₁ -C₃ alkyl.

5! Another embodiment of this invention is the reagent of embodiment 4!wherein:

Q represents a biologically active molecule selected from the group:IIb/IIIa receptor antagonists and chemotactic peptides;

d' is 1;

L_(n) is:

--(CR⁵⁵ R⁵⁶)_(g") -- Y¹ (CR⁵⁵ R⁵⁶)_(f) Y² !_(f') --(CR⁵⁵ R⁵⁶)_(g") --,

wherein:

g" is 0-5;

f is 0-5;

f' is 1-5;

Y¹ and Y², at each occurrence, are independently selected from:

O, NR⁵⁶, C═O, C(═O)O, OC(═O)O, C(═O)NH--, C═NR⁵⁶, S, NHC(═O), (NH)₂C(═O), (NH)₂ C═S;

R⁵⁵ and R⁵⁶ are hydrogen;

H_(z) is a stable hydrazone of formula: ##STR5## wherein, R⁴⁰ isindependently selected at each occurrence from the group: heterocyclesubstituted with R⁵² ;

R⁴¹ is hydrogen;

R⁵² is a bond to L_(n) ;

R⁸⁰ is independently selected from the group:

--CO₂ R⁸⁵ ;

C₂ -C₃ 1-alkene substituted with 0-1 R⁸⁴ ;

aryl substituted with 0-1 R⁸⁴ ;

unsaturated heterocycle substituted with 0-1 R⁸⁴ ;

R⁸¹ is H;

R⁸⁴ is independently selected at each occurrence from the group:

--CO₂ R⁸⁵ ;

--OR⁸⁵ ;

--SO₃ H;

--N(R⁸⁵)₂ ;

R⁸⁵ is independently selected at each occurrence from the group:

H and methyl.

6! Another embodiment of this invention are the reagents of embodiment3! that are: ##STR6## 7! Another embodiment of this invention is a kitfor preparing a radiopharmaceutical comprising:

(a) a predetermined quantity of a sterile, pharmaceutically acceptablereagent of any of claims 1-6;

(b) a predetermined quantity of one or more sterile, pharmaceuticallyacceptable ancillary ligand(s);

(c) a predetermined quantity of a sterile, pharmaceutically acceptablereducing agent; and

(d) optionally, a predetermined quantity of a sterile, pharmaceuticallyacceptable component selected from the group: transfer ligands, buffers,lyophilization aids, stabilization aids, solubilization aids andbacteriostats.

8! Another embodiment of this invention is a kit for preparing aradiopharmaceutical comprising:

(a) a predetermined quantity of a sterile, pharmaceutically acceptablereagent of any of claims 1-6;

(b) a predetermined quantity of two sterile, pharmaceutically acceptableancillary ligand(s);

(c) a predetermined quantity of a sterile, pharmaceutically acceptablereducing agent; and

(d) optionally, a predetermined quantity of a sterile, pharmaceuticallyacceptable component selected from the group:

transfer ligands, buffers, lyophilization aids, stabilization aids,solubilization aids and bacteriostats.

9! Another embodiment of this invention is a stable hydrazone compounduseful for the synthesis of the reagents of embodiment 1-6! having theformula:

    R.sup.44 (C═O).sub.s (R.sup.45)N--N═CR.sup.8 OR.sup.81

wherein:

s is 0 or 1;

R⁴⁴ is selected from the group: aryl substituted with 1 R⁵⁹ ; andheterocycle substituted with 1 R⁵⁹ ;

R⁴⁵ is selected from the group: hydrogen and C₁ -C₆ alkyl,

R⁵⁹ is a chemically reactive moiety selected from the group:

alkyl substituted with halogen;

acid anhydride;

acid halide;

active ester;

isothiocyanate;

maleimide;

R⁸⁰ and R⁸¹ are independently selected from the group:

H;

C₁ -C₁₀ alkyl;

--CN;

--CO₂ R⁸⁵ ;

--C(═O)R⁸⁵ ;

--C(═O)N(R⁸⁵)₂ ;

C₂ -C₁₀ 1-alkene substituted with 0-3 R⁸⁴ ;

C₂ -C₁₀ 1-alkyne substituted with 0-3 R⁸⁴ ;

aryl substituted with 0-3 R⁸⁴ ;

unsaturated heterocycle substituted with 0-3 R⁸⁴ ; and

unsaturated carbocycle substituted with 0-3 R⁸⁴ ;

provided that when one of R⁸⁰ and R⁸¹ is H or alkyl, then the other isnot H or alkyl;

or, alternatively, R⁸⁰ and R⁸¹, may be taken together with the showndivalent carbon radical to form: ##STR7## wherein: R⁸² and R⁸³ areindependently selected at each occurrence from the group:

H;

R⁸⁴ ;

C₁ -C₁₀ alkyl substituted with

0--3 R⁸⁴ ;

C₂ -C₁₀ alkenyl substituted with 0-3 R⁸⁴ ;

C₂ -C₁₀ alkynyl substituted with 0-3 R⁸⁴ ;

aryl substituted with 0-3 R⁸⁴ ;

heterocycle substituted with 0-3 R⁸⁴ ; and

carbocycle substituted with 0-3 R⁸⁴ ;

or, alternatively,

R⁸², R⁸³ may be taken together to form a fused aromatic or heterocyclicring; and

a and b indicate the positions of optional double bonds;

n is 0 or 1,

R⁸⁴ is independently selected at each occurrence from the group: ═O, F,Cl, Br, I, --CF₃, --CN, --CO₂ R⁸⁵, --C(═O)R⁸⁵, --C(═O)N(R⁸⁵)₂, --CH₂OR⁸⁵, --OC(═O)R⁸⁵, --OC (═O) OR^(85a), --OR⁸⁵, --OC(═O)N(R⁸⁵)₂, --NR⁸⁵C(═O) R⁸⁵, --NR⁸⁶ C(═O)OR^(85a), --NR⁸⁵ C(═O)N(R⁸⁵)₂, --SO₃ Na, --NR⁸⁶SO₂ N(R⁸⁵)₂, --NR⁸⁶ SO₂ R^(85a), --SO₃ H, --SO₂ R^(85a), --SR⁸⁵,--S(═O)R^(85a), --SO₂ N(R⁸⁵)₂, --N(R⁸⁵)₂, N(R⁸⁵)₃ ⁺, --NHC(═NH)NHR⁸⁵,--C(═NH)NHR⁸⁵, ═NOR⁸⁵, --C(═O)NHOR⁸⁵, --OCH₂ CO₂ H,2-(1-morpholino)ethoxy;

R⁸⁵, R^(85a), and R⁸⁶ are independently selected at each occurrence fromthe group: hydrogen, C₁ -C₆ alkyl.

10! Another embodiment of this invention is the compound of embodiment9! wherein:

s=0;

R⁵⁹ is selected from the group: ##STR8## R⁸⁰ is independently selectedfrom the group: --CO₂ R⁸⁵ ;

C_(2-C) ₅ 1-alkene substituted with 0-3 R⁸⁴ ;

C₂ -C₅ 1-alkyne substituted with 0-3 R⁸⁴ ;

aryl substituted with 0-3 R⁸⁴ ;

unsaturated heterocycle substituted with 0-3 R⁸⁴ ;

R⁸¹ is independently selected from the group: H and C₁ -C₅ alkyl;

or, alternatively, R⁸⁰ and R⁸¹, may be taken together with the showndivalent carbon radical to form ##STR9## wherein R⁸² and R⁸³ may beindependently selected from the group: H and R⁸⁴ ;

or, alternatively, R⁸², R⁸³ may be taken together to form a fusedaromatic or heterocyclic ring; and

a and b indicate the positions of optional double bonds;

n is 0 or 1,

R⁸⁴ is independently selected at each occurrence from the group: --CO₂R⁸⁵, --C(═O)N(R⁸⁵)₂, --CH₂ OR⁸⁵, --OC(═O)R⁸⁵, --OR⁸⁵, --SO₃ H, --SO₃ Na,--N(R⁸⁵)₂, --OCH₂ CO₂ H;

R⁸⁵ is independently selected at each occurrence from the group:hydrogen and C₁ -C₃ alkyl.

11! Another embodiment of this invention is the compound of embodiment10! wherein:

R⁸⁰ is independently selected from the group:

--CO₂ R⁸⁵ ;

C₂ -C₃ 1-alkene substituted with 0-1 R⁸⁴ ;

aryl substituted with 0-1 R⁸⁴ ;

unsaturated heterocycle substituted with 0-1 R⁸⁴ ;

R⁸¹ is H;

R⁸⁴ is independently selected at each occurrence from the group: --CO₂R⁸⁵, --OR⁸⁵, --SO₃ H, --SO₃ Na, --N(R⁸⁵)₂ ;

R⁸⁵ is independently selected at each occurrence from the group:

H and methyl.

12! Another embodiment of this invention are the compounds of embodiment9! that are: ##STR10##

When any variable occurs more than one time in any constituent or in anyformula, its definition on each occurrence is independent of itsdefinition at every other occurrence. Thus, for example, if a group isshown to be substituted with 0-2 R⁵², then said group may optionally besubstituted with up to two R⁵² and R⁵² at each occurrence is selectedindependently from the defined list of possible R⁵². Also, by ofexample, for the group --N(R⁵³)₂, each of the two R⁵³ substituents on Nis independently selected from the defined list of possible R⁵³.Combinations of substituents and/or variables are permissible only ifsuch combinations result in stable compounds.

By "stable compound" or "stable structure" is meant herein a compoundthat is sufficiently robust to survive isolation to a useful degree ofpurity from a reaction mixture, and formulation into an efficaciousdiagnostic agent.

The term "hydrazone", as used herein, means that the moiety, group orcompound so described is comprised of at least one divalent carbonradical (or methylene group) bound to a nitrogen atom on a hydrazine orhydrazide through a double bond.

The term "substituted", as used herein, means that one or more hydrogenson the designated atom or group is replaced with a selection from theindicated group, provided that the designated atom's or group's normalvalency is not exceeded, and that the substitution results in a stablecompound. When a substituent is keto (i.e., ═O), then 2 hydrogens on theatom are replaced.

The term "bond", as used herein, means either a single, double or triplebond.

As used herein, "alkyl" is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms; "cycloalkyl" or "carbocycle" isintended to include saturated ring groups, including mono-,bi- orpoly-cyclic ring systems, such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl and adamantyl; "bicycloalkyl" isintended to include saturated bicyclic ring groups such as3.3.0!bicyclooctane, 4.3.0!bicyclononane, 4.4.0!bicyclodecane (decalin),2.2.2!bicyclooctane, and so forth;

As used herein, the term "alkene" or "alkenyl" is intended to includeboth branched and straight-chain groups of the formula C_(n) H_(2n-1)having the specified number of carbon atoms. The term "1-alkene" or"1-alkenyl" means that the double bond is between the first and secondcarbon atoms from the point of attachment.

As used herein, the term "alkyne" or "alkynyl" is intended to includeboth branched and straight-chain groups of the formula C_(n) H_(2n-3)having the specified number of carbon atoms. The term "1-alkyne" or"1-alkynyl" means that the triple bond is between the first and secondcarbon atoms from the point of attachment.

As used herein, "aryl" or "aromatic residue" is intended to mean phenylor naphthyl, which when substituted, the substitution can be at anyposition.

As used herein, the term "heterocycle" or "heterocyclic ring system" isintended to mean a stable 5- to 7- membered monocyclic or bicyclic or 7-to 10-membered bicyclic heterocyclic ring which may be saturated,partially unsaturated, or aromatic, and which consists of carbon atomsand from 1 to 4 heteroatoms selected independently from the groupconsisting of N, O and S and wherein the nitrogen and sulfur heteroatomsmay optionally be oxidized, and the nitrogen may optionally bequaternized, and including any bicyclic group in which any of theabove-defined heterocyclic rings is fused to a benzene ring. Theheterocyclic ring may be attached to its pendant group at any heteroatomor carbon atom which results in a stable structure. The heterocyclicrings described herein may be substituted on carbon or on a nitrogenatom if the resulting compound is stable. Examples of such heterocyclesinclude, but are not limited to, benzopyranyl, thiadiazine, tetrazolyl,benzofuranyl, benzothiophenyl, indolene, quinoline, isoquinolinyl orbenzimidazolyl, piperidinyl, 4-piperidone, 2-pyrrolidone,tetrahydrofuran, tetrahydroquinoline, tetrahydroisoquinoline,decahydroquinoline, octahydroisoquinoline, azocine, triazine (including1,2,3-, 1,2,4-, and 1,3,5-triazine), 6H-1,2,5-thiadiazine,2H,6H-1,5,2-dithiazine, thiophene, tetrahydrothiophene, thianthrene,furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin,2H-pyrrole, pyrrole, imidazole, pyrazole, thiazole, isothiazole, oxazole(including 1,2,4- and 1,3,4-oxazole), isoxazole, triazole, pyridine,pyrazine, pyrimidine, pyridazine, indolizine, isoindole, 3H-indole,indole, 1H-indazole, purine, 4H-quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, 4aH-carbazole, carbazole, β-carboline, phenanthridine,acridine, perimidine, phenanthroline, phenazine, phenarsazine,phenothiazine, furazan, phenoxazine, isochroman, chroman, pyrrolidine,pyrroline, imidazolidine, imidazoline, pyrazolidine, pyrazoline,piperazine, indoline, isoindoline, quinuclidine, or morpholine. Alsoincluded are fused ring and spiro compounds containing, for example, theabove heterocycles.

As used herein to describe the substituents R⁸⁰ and R⁸¹, the term"unsaturated carbocycle" means a carbocycle that has at least onemultiple bond, that one multiple bond being between the carbon atomattached to the divalent carbon radical specified in the formula of thestable hydrazone moiety and an adjacent carbon atom.

As used herein to describe the substituents R⁸⁰ and R⁸¹, the term"unsaturated heterocycle" means a heterocycle that has at least onemultiple bond, that one multiple bond being between the carbon atomattached to the divalent carbon radical specified in the formula of thestable hydrazone moiety and an adjacent carbon atom. An aromaticheterocycle is considered an unsaturated heterocycle.

The term "salt", as used herein, is used as defined in the CRC Handbookof Chemistry and Physics, 65th Edition, CRC Press, Boca Raton, Fla.,1984, as any substance which yields ions, other than hydrogen orhydroxyl ions.

A "reducing agent" is a compound that reacts with the radionuclide,which is typically obtained as a relatively unreactive, high oxidationstate compound, to lower its oxidation state by transfering electron(s)to the radionuclide, thereby making it more reactive. Reducing agentsuseful in the preparation of radiopharmaceuticals and in diagnostic kitsuseful for the preparation of said radiopharmaceuticals include but arenot limited to stannous chloride, stannous fluoride, formamidinesulfinic acid, ascorbic acid, cysteine, phosphines, and cuprous orferrous salts. Other reducing agents are described in Brodack et. al.,PCT Application 94/22496, which is incorporated herein by reference.

A "transfer ligand" is a ligand that forms an intermediate complex withthe radionuclide that is stable enough to prevent unwantedside-reactions but labile enough to be converted to theradiopharmaceutical. The formation of the intermediate complex iskinetically favored while the formation of the radiopharmaceutical isthermodynamically favored. Transfer ligands useful in the preparation ofradiopharmaceuticals and in diagnostic kits useful for the preparationof said radiopharmaceuticals include but are not limited to gluconate,glucoheptonate, mannitol, glucarate,N,N,N',N'-ethylenediaminetetraacetic acid, pyrophosphate andmethylenediphosphonate. In general, transfer ligands are comprised ofoxygen or nitrogen donor atoms.

The term "donor atom" refers to the atom directly attached to a metal bya chemical bond.

"Ancillary" or "co-ligands" are ligands that are incorporated into theradiopharmaceutical during its synthesis. They serve to complete thecoordination sphere of the radionuclide together with the chelator orradionuclide bonding unit of the reagent. For radiopharmaceuticalscomprised of a binary ligand system, the radionuclide coordinationsphere is composed of one or more chelators or bonding units from one ormore reagents and one or more ancillary or co-ligands, provided thatthere are a total of two types of ligands, chelators or bonding units.For example, a radiopharmaceutical comprised of one chelator or bondingunit from one reagent and two of the same ancillary or co-ligands and aradiopharmaceutical comprised of two chelators or bonding units from oneor two reagents and one ancillary or co-ligand are both considered to becomprised of binary ligand systems. For radiopharmaceuticals comprisedof a ternary ligand system, the radionuclide coordination sphere iscomposed of one or more chelators or bonding units from one or morereagents and one or more of two different types of ancillary orco-ligands, provided that there are a total of three types of ligands,chelators or bonding units. For example, a radiopharmaceutical comprisedof one chelator or bonding unit from one reagent and two differentancillary or co-ligands is considered to be comprised of a ternaryligand system. Co-pending application U.S. Ser. No. 08/415,908,908,which is incorporated herein by reference, discloses and teachesancillary ligands.

A "chelator" or "bonding unit" is the moiety or group on a reagent thatbinds to a metal radionuclide through the formation of chemical bondswith one or more donor atoms.

The term "binding site" means the site in vivo that binds a biologicallyactive molecule.

Ancillary or co-ligands useful in the preparation ofradiopharmaceuticals and in diagnostic kits useful for the preparationof said radiopharmaceuticals are comprised of one or more oxygen,nitrogen, carbon, sulfur, phosphorus, arsenic, selenium, and telluriumdonor atoms. A ligand can be a transfer ligand in the synthesis of aradiopharmaceutical and also serve as an ancillary or co-ligand inanother radiopharmaceutical. Whether a ligand is termed a transfer orancillary or co-ligand depends on whether the ligand remains in theradionuclide coordination sphere in the radiopharmaceutical, which isdetermined by the coordination chemistry of the radionuclide and thechelator or bonding unit of the reagent or reagents.

A "diagnostic kit" comprises a collection of components, termed theformulation, in one or more vials which are used by the practising enduser in a clinical or pharmacy setting to synthesize theradiopharmaceutical. The kit provides all the requisite components tosynthesize and use the radiopharmaceutical except those that arecommonly available to the practising end user, such as water or salinefor injection, a solution of the radionuclide, equipment for heating thekit during the synthesis of the radiopharmaceutical if required,equipment necessary for administering the radiopharmaceutical to thepatient such as syringes and shielding, and imaging equipment.

A "buffer" is a compound that is used to control the pH of the kitduring its manufacture and during the synthesis of theradiopharmaceutical.

A "lyophilization aid" is a component that has favorable physicalproperties for lyophilization, such as the glass transition temperature,and is added to the diagnostic kit to improve the physical properties ofthe combination of all the components of the kit for lyophilization.

A "stabilization aid" is a component that is added to theradiopharmaceutical or to the diagnostic kit either to stabilize theradiopharmaceutical once it is synthesized or to prolong the shelf-lifeof the kit before it must be used. Stabilization aids can beantioxidants, reducing agents or radical scavengers and can provideimproved stability by reacting preferentially with species that degradeother components or the radiopharmaceutical.

A "solubilization aid" is a component that improves the solubility ofone or more other components in the medium required for the synthesis ofthe radiopharmaceutical.

A "bacteriostat" is a component that inhibits the growth of bacteria inthe diagnostic kit either during its storage before use of after the kitis used to synthesize the radiopharmaceutical.

The following abbreviations are used in this application:

    ______________________________________                                        Acm           acetamidomethyl                                                 D-Abu         D-2-aminobutyric acid                                           5-Aca         5-aminocaproamide (5-                                                         aminohexanamide)                                                b-Ala, b-Ala or                                                                             3-aminopropionic acid                                           bAla                                                                          Boc           t-butyloxycarbonyl                                              Boc-iodo-Mamb t-butyloxycarbonyl-3-aminomethyl-4-                             iodo-         benzoic acid                                                    Boc-Mamb      t-butyloxycarbonyl-3-                                                         aminomethylbenzoic acid                                         Boc-ON         2-(tert-                                                                     butyloxycarbonyloxylimino)-2-                                                 phenylacetonitrile                                              Cl.sub.2 Bzl  dichlorobenzyl                                                  CBZ, Cb or Z  Carbobenzyloxy                                                  DCC           dicyclohexylcarbodiimide                                        DIEA          diisopropylethylamine                                           di-NMeOrn     N-aMe-N-gMe-ornithine                                           DMAP          4-dimethylaminopyridine                                         HBTU          2-(1H-Benzotriazol-1-yl)-1,1,3,3-                                             tetramethyluronium                                                            hexafluorophosphate                                             Hynic         hydrazinonicotinyl                                              NMeArg or     a-N-methyl arginine                                             MeArg                                                                         NMeAmf        N-Methylaminomethylphenylalanine                                NMeAsp        a-N-methyl aspartic acid                                        NMeGly or     N-methyl glycine                                                MeGly                                                                         NMe-Mamb      N-methyl-3-aminomethylbenzoic acid                              NMM           N-methylmorpholine                                              OcHex         O-cyclohexyl                                                    OBzl          O-benzyl                                                        oSu           O-succinimidyl                                                  pNP           p-nitrophenyl                                                   TBTU          2-(1H-Benzotriazol-1-yl)-1,1,3,3-                                             tetramethyluronium                                                            tetrafluoroborate                                               Teoc          2-(Trimethylsilyl)ethyloxycarbonyl                              Tos           tosyl                                                           TPPTS         tris(3-sulfonatophenyl)phosphine                                              trisodium salt                                                  Tr            trityl                                                          ______________________________________                                    

The following conventional three-letter amino acid abbreviations areused herein; the conventional one-letter amino acid abbreviations arenot used herein:

Ala=alanine

Arg=arginine

Asn=asparagine

Asp=aspartic acid

Cys=cysteine

Gln=glutamine

Glu=glutamic acid

Gly=glycine

His=histidine

Ile=isoleucine

Leu=leucine

Lys=lysine

Met=methionine

Nle=norleucine

Phe=phenylalanine

Phg=phenylglycine

Pro=proline

Ser=serine

Thr=threonine

Trp=tryptophan

Tyr=tyrosine

Val=valine

The biologically active molecule Q can be a protein, antibody, antibodyfragment, peptide or polypeptide, or peptidomimetic that is comprised ofa recognition sequence or unit for a receptor or binding site expressedat the site of the disease, or for a receptor or binding site expressedon platelets or leukocytes. The exact chemical composition of Q isselected based on the disease state to be diagnosed, the mechanism oflocalization to be utilized, and to provide an optimium combination ofrates of localization, clearance and radionuclidic decay.

For the purposes of this invention, the term thromboembolic disease istaken to include both venous and arterial disorders and pulmonaryembolism, resulting from the formation of blood clots.

For the diagnosis of thromboembolic disorders or atherosclerosis, Q isselected from the group including the cyclic IIb/IIIa receptorantagonist compounds described in co-pending U.S. Ser. No.08/415,908,861 (equivalent to WO 94/22494); the RGD containing peptidesdescribed in U.S. Pat. Nos. 4,578,079, 4,792,525, the applications PCTUS88/04403, PCT US89/01742, PCT US90/03788, PCT US91/02356 and by Ojimaet. al., 204th Meeting of the Amer. Chem. Soc., 1992, Abstract 44; thepeptides that are fibrinogen receptor antagonists described in EuropeanPatent Applications 90202015.5, 90202030.4, 90202032.2, 90202032.0,90311148.2, 90311151.6, 90311537.6, the specific binding peptides andpolypeptides described as lIb/IIIa receptor ligands, ligands for thepolymerization site of fibrin, laminin derivatives, ligands forfibrinogen, or thrombin ligands in PCT WO 93/23085 (excluding thetechnetium binding groups); the oligopeptides that correspond to theIIIa protein described in PCT WO90/00178; the hirudin-based peptidesdescribed in PCT WO90/03391; the IIb/IIIa receptor ligands described inPCT WO90/15818; the thrombus, platelet binding or atherosclerotic plaquebinding peptides described in PCT WO92/13572 (excluding the technetiumbinding group) or GB 9313965.7; the fibrin binding peptides described inU.S. Pat. Nos. 4,427,646 and 5,270,030; the hirudin-based peptidesdescribed in U.S. Pat. No. 5,279,812; or the fibrin binding proteinsdescribed in U.S. Pat. No. 5,217,705; the guanine derivatives that bindto the IIb/IIIa receptor described in U.S. Pat. No. 5,086,069; or thetyrosine derivatives described in European Patent Application 0478328A1,and by Hartman et. al., J. Med. Chem., 1992, 35, 4640; or oxidized lowdensity lipoprotein (LDL).

For the diagnosis of infection, inflammation or transplant rejection, Qis selected from the group including the leukocyte binding peptidesdescribed in PCT WO93/17719 (excluding the technetium binding group),PCT WO92/13572 (excluding the technetium binding group) or U.S. Ser. No.08-140000; the chemotactic peptides described in Eur. Pat. Appl.90108734.6 or A. Fischman et. al., Semin. Nuc. Med., 1994, 24, 154; orthe leukostimulatory agents described in U.S. Pat. No. 5,277,892.

For the diagnosis of cancer, Q is selected from the group ofsomatostatin analogs described in UK Application 8927255.3 or PCTWO94/00489, the selectin binding peptides described in PCT WO94/05269,the biological-function domains described in PCT WO93/12819, PlateletFactor 4 or the growth factors (PDGF, EGF, FGF, TNF, MCSF or I11-8).

Q may also represent proteins, antibodies, antibody fragments, peptides,polypeptides, or peptidomimetics that bind to receptors or binding siteson other tissues, organs, enzymes or fluids. Examples include theβ-amyloid proteins that have been demonstrated to accumulate in patientswith Alzheimer's disease, atrial naturetic factor derived peptides thatbind to myocardial and renal receptors, antimyosin antibodies that bindto areas of infarcted tissues, or nitroimidazole derivatives thatlocalize in hypoxic areas in vivo.

The reagents of the present invention are comprised of a biologicallyactive group, Q, attached to a stable hydrazone, H_(z), and optionallycomprising a linking group, L_(n), between said biologically activegroup and said stable hydrazone. The stable hydrazone is a protectedform of a hydrazine or hydrazide chelator or bonding unit, designated asC_(h) in co-pending U.S. Ser. No. 08/415,908,861, that is eitherdirectly attached to the moiety Q or is attached to the linking groupL_(n) which is attached to Q. The chelator or bonding unit becomes boundto the radionuclide (and is designated C_(h') in the bound state in U.S.Ser. No. 08/415,908,908) in the radiopharmaceuticals synthesized usingthe reagents of the present invention.

The substituents R⁸⁰ and R⁸¹ of this invention are chosen to improve thestability of the hydrazone over that achievable using substituentscomprised solely of hydrogen or lower alkyl. The improved stability isnecessary because hydrazones in which the substituents are solelyhydrogen or lower alkyl are reactive with other aldehydes and ketones, anumber of which are commonly found in a pharmaceutical manufacturingsetting. A particularly ubiquitous aldehyde is formaldehyde, which iscommonly used in disinfectants. The reaction of a lower alkyl hydrazonewith an aldehyde or ketone can proceed as shown in Scheme 1. ##STR11##

The stable hydrazone protected reagents of this invention may becommercialized as radiopharmaceutical precursers. Lower alkyl protectedhydrazones can not be so commercialized, due to their inherentinstability. If the lower alkyl hydrazone depicted in Scheme 1 is partof a reagent for the preparation of a radiopharmaceutical, when itundergoes the shown reaction with other aldehydes and ketones it will bedecomposed or degraded into one or more other hydrazones depending onthe number of aldehydes and ketones with which it comes into contact.These decomposition products constitute impurities in the reagent whichmust be minimized or avoided. The elimination of all aldehydes andketones is very difficult in a manufacturing setting because they can beextracted from a number of the materials, particularly plastics andrubber stoppers, used in the manufacturing process and are present inthe common disinfectants. The use of stable hydrazones in the novelreagents of the present invention, which are comprised of stablehydrazone modified biologically active molecules, obviates this problem.Thus, the stable hydrazone reagents of this invention enjoy asignificant advantage over the lower alkyl protected hydrazonesdisclosed in the prior art, this due to the increased stability of thestable hydrazone reagents, which renders them capable ofcommercialization.

The stable hydrazone group, H_(z), of the formula --N(R⁴⁰ R⁴¹)N═C(R⁸⁰R⁸¹), differs from the lower alkyl hydrazones in that one of thesubstituents R⁸⁰ and R⁸¹ is selected from the group: nitrile, carboxylicacids, carboxylic acid esters, carboxamides, 1-alkenes, 1-alkynes, aryl,unsaturated heterocycle, and unsaturated carbocycle; or the twosubstituents R⁸⁰ and R⁸¹ are taken together to form a ring system. Thesubstituents in the group serve to stabilize the hydrazone by providinga conjugated π system either as a carbon-carbon double bond, acarbon-oxygen double bond, a carbon-carbon triple bond, acarbon-nitrogen triple bond or an aromatic ring. Stability can also beprovided by the chelate effect if the substituents R⁸⁰ and R⁸¹ are takentogether to form a ring system.

The reagents of the present invention can be synthesized by a variety ofmethods. The hydrazine and hydrazide precursers may be prepared asdescribed in co-pending application U.S. Ser. No. 08/415,908,861. Thestable hydrazone group H_(z) can be introduced at any step in thesynthesis of the reagent provided that it is stable to subsequentreaction conditions. One synthetic approach involves the reaction of astable hydrazone group bearing a coupling functionality with abiologically active molecule, Q, optionally bearing a linking group,L_(n). A coupling functionality is a chemically reactive moiety capableof reacting with a biologically active molecule, optionally bearing alinking group, to bind the stable hydrazone thereto. For biologicallyactive molecules bearing a linking group, the stable hydrazone is boundto the linking group.

The reaction of the stable hydrazone bearing a chemically reactivemoiety with a biologically active molecule or linker modifiedbiologically active molecule can be performed by direct combination ofthe two reactants in a suitable solvent and under suitable reactionconditions. A solvent or reaction condition is suitable if the stablehydrazone-biologically active molecule or stablehydrazone-linker-biologically active molecule reagent is formed withouta significant loss of biological activity due to the use of said solventor condition.

Examples of chemically reactive moieties include an alkyl group bearinga good leaving group such as a halide, a carbonyl group such as an acidanhydride, acid halide, or active ester, an isothiocyanate orsubstituted isothiocyanate, or a maleimide. An active ester is an esterthat is more reactive in nucleophilic substitution reactions such astetrafluorophenyl, N-succinimidyl, and nitrophenyl. In the reactionbetween the stable hydrazone bearing a chemically reactive moiety and abiologically active molecule or linker modified biologically activemolecule, either reactant can serve as the nucleophile. More detaileddescriptions of these coupling reactions can be found in Brinkley, M.,Bioconjugate Chemistry, 1992, Vol. 3, No. 1, which is incorporatedherein by reference. Also, U.S. Pat. No. 5,206,370, which isincorporated herein, discloses other examples of chemically reactivemoieties.

Another synthetic approach involves formation of the stable hydrazone asthe last step in the synthesis of the reagent. Compounds of the formula(Q)_(d') --L_(n) --C_(h), in which C_(h) is --R⁴⁰ R⁴¹ NNH₂, thesynthesis of which are described in co-pending U.S. Ser. No.08/415,908,861, can be reacted with carbonyl-containing compounds of theformula R⁸ C(═O)R⁸¹ in a suitable solvent under suitable reactionconditions. A solvent or reaction condition is suitable if the reagentis formed without a significant loss of biological activity due to theuse of said solvent or condition.

Stable hydrazones bearing a chemically reactive group useful in thesynthesis of the reagents of the present invention can be synthesized asshown in Scheme 2. ##STR12## Hydrazinonicotinic acid is reacted with acarbonyl-containing compound, R⁸⁰ C(═O)R⁸¹, in dimethylformamide to formthe respective stable hydrazone of nicotinic acid. Reaction of thestable hydrazone solution with N-hydroxysuccinimide (NHS) in thepresence of dicyclohexylcarbodiimide (DCC) results in the succinimidylester of the respective stable hydrazone of nicotinic acid. Thesyntheses of specific stable hydrazones bearing a succinimidyl esterchemically reactive moiety are described in the Example Section.

The stable hydrazones bearing a succinimidyl ester moiety can be used toprepare reagents of the present invention by reaction with an aminogroup on a biologically active molecule or linker-modified biologicallyactive molecule to form an amide bond. The synthesis of reagents byreaction with linker modified cyclic IIb/IIIa receptor antagonists isshown in Scheme 3. ##STR13## A dimethylformamide solution of a hydrazonebearing a succinimidyl ester moiety is combined with the linker modifiedcyclic IIb/IIIa receptor antagonistcyclo-(D-Val-NMeArg-Gly-Asp-Mamb(5-Aca)) dissolved in DMF to form areagent, cyclo-(D-Val-NMeArg-Gly-Asp-Mamb(H_(z) -5-Aca)).Cyclo-(D-Val-NMeArg-Gly-Asp-Mamb(5-Aca)) is synthesized as described incopending U.S. Ser. No. 08/415,908,861. The crude reagent can bepurified by preparative high performance liquid chromatography (HPLC) orby a number of other methods known to those skilled in the art, such asrecrystallization, column chromatography and solvent extraction.

An alternative approach for the synthesis of the reagent of the presentinvention involves reaction of a carbonyl-containing compound of theformula R⁸⁰ C(═O)R⁸¹ with a compound of the formula (Q)_(d) '--L_(n)--C_(h), wherein C_(h) is --R⁴⁰ R⁴¹ NNH₂, as shown in Scheme 4.##STR14## The cyclic IIb/IIIA receptor antagonist,cyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hynic-5-Aca)), which can be synthesizedas described in co-pending U.S. Ser. No. 08/415,908,861, is reacted witha carbonyl-containing compound of the formula R⁸⁰ (C═O)R⁸¹ indimethylformamide to give a reagentcyclo-(D-Val-NMeArg-Gly-Asp-Mamb(H_(z) -5-Aca)). The crude reagent canbe purified by preparative high performance liquid chromatography (HPLC)or by a number of other methods known to those skilled in the art, suchas recrystallization, column chromatography and solvent extraction.

The reagents of the present invention of the formula (Q)_(d') --L_(n)--H_(z), are useful for the preparation of radiopharmaceuticalsdisclosed in copending patent application Ser. No. 08/415,908,908 offormula:

     (Q).sub.d' --L.sub.n --C.sub.h' !.sub.x --M.sub.t (A.sub.L1).sub.y (A.sub.L2).sub.z                                          (2),

wherein Q, d', and L_(n) are as defined above and C_(h') is aradionuclide metal chelator or bonding unit bound to the transitionmetal radionuclide, M_(t), of the formulae R⁴⁰ N═N⁺ ═, R⁴⁰ R⁴¹ N--N═,R⁴⁰ N═, or R⁴⁰ N═N(H)--, A_(L1) is a first ancillary or co-ligand,A_(L2) is a second ancillary or co-ligand, x and y are independently 1or 2, and z is independently an integer from 0 to 4. The transitionmetal radionuclide, M_(t), can be selected from the group:technetium-99m, rhenium-186 and rhenium-188.

The group C_(h') is termed a hydrazido (of formula R⁴⁰ R⁴¹ N--N═),diazenido (of formula R⁴⁰ N═N⁺ ═ or R⁴⁰ N═N(H)--) or imido (of formulaR⁴⁰ N═) group and serves as the point of attachment of the radionuclideto the remainder of the radiopharmaceutical designated by the formula(Q)_(d') --L_(n). A diazenido group can be either terminal (only oneatom of the group is bound to the radionuclide) or chelating. In orderto have a chelating diazenido group at least one other atom of thegroup, located on R⁴⁰, must also be bound to the radionuclide. The atomsbound to the metal are termed donor atoms. The hydrazido and imidogroups are exclusively terminal.

The coordination sphere of the radionuclide includes all the ligands orgroups bound to the radionuclide. For a transition metal radionuclide,M_(t), to be stable it typically has a coordination number comprised ofan integer greater than or equal to 5 and less than or equal to 7; thatis there are 5 to 7 atoms bound to the metal and it is said to have acomplete coordination sphere. If the chelator or bonding unit C_(h')does not provide all of the atoms necessary to stabilize the metalradionuclide by completing its coordination sphere, the coordinationsphere is completed by donor atoms from other ligands, termed ancillaryor co-ligands, which can also be either terminal or chelating.

A large number of ligands can serve as ancillary or co-ligands, thechoice of which is determined by a variety of considerations such as theease of synthesis of the radiopharmaceutical, the chemical and physicalproperties of the ancillary ligand, the rate of formation, the yield,and the number of isomeric forms of the resulting radiopharmaceuticals,the ability to administer said ancillary or co-ligand to a patientwithout adverse physiological consequences to said patient, and thecompatibility of the ligand in a lyophilized kit formulation. The chargeand lipophilicity of the ancillary ligand will effect the charge andlipophilicity of the radiopharmaceuticals. For example, the use of4,5-dihydroxy-1,3-benzene disulfonate results in radiopharmaceuticalswith an additional two anionic groups because the sulfonate groups willbe anionic under physiological conditions. The use of N-alkylsubstituted 3,4-hydroxypyridinones results in radiopharmaceuticals withvarying degrees of lipophilicity depending on the size of the alkylsubstituents.

The radiopharmaceuticals prepared from the reagents of the presentinvention can be comprised of one or two ancillary or co-ligands,designated A_(L1), in a binary ligand system. The one or two ancillaryor co-ligands, A_(L1), comprising the radiopharmaceuticals can beindependently selected from the group: dioxygen ligands, functionalizedaminocarboxylates and halides; provided that the coordination sphere ofthe radionuclide is complete.

Ancillary dioxygen ligands include ligands that coordinate to the metalion through at least two oxygen donor atoms. Examples include but arenot limited to: glucoheptonate, gluconate, 2-hydroxyisobutyrate,lactate, tartrate, mannitol, glucarate, maltol, Kojic acid,2,2-bis(hydroxymethyl)propionic acid, 4,5-dihydroxy-1,3-benzenedisulfonate, or substituted or unsubstituted 1,2 or 3,4hydroxypyridinones, or pharmaceutically acceptable salts thereof.

Functionalized aminocarboxylates include ligands that have a combinationof nitrogen and oxygen donor atoms. Examples include but are not limitedto: iminodiacetic acid, 2,3 diaminopropionic acid, nitrilotriaceticacid, N,N'-ethylenediamine diacetic acid, N,N,N'-ethylenediaminetriacetic acid, hydroxyethylethylenediamine triacetic acid,N,N'-ethylenediamine bis-hydroxyphenylglycine, or the ligands describedin Eur. Pat. Appl. 93302712.0, or pharmaceutically acceptable saltsthereof.

Halides can be chloride, bromide, fluoride or iodide or pharmaceuticallyacceptable salts thereof.

Of particular utility are radiopharmaceuticals prepared from thereagents of the present invention comprised of two different types ofancillary or co-ligands, one or two ligands designated the firstancillary or co-ligand or ligands, A_(L1), and independently selectedfrom the group: dioxygen ligands, functionalized aminocarboxylates andhalides; and one to four ligands designated the second ancillary orco-ligand or ligands, A_(L2), selected from the group: trisubstitutedphosphines, trisubstituted arsines, tetrasubstituted diphosphines andtetrasubstituted diarsines, in a ternary ligand system. We havedisclosed in copending U.S. Ser. No. 08/415,908, thatradiopharmaceuticals of the formula (Q)_(d') L_(n) --C_(h') !_(x)--M_(t) (A_(L1))_(y) (A_(L2))_(z) comprised of one or more ancillary orco-ligands A_(L2) are stable compared to said radiopharmaceuticals thatare not comprised of one or more ancillary ligands, A_(L2) ; that is,they have a minimal number of isomeric forms, the relative ratios ofwhich do not change significantly with time, and that remainsubstantially intact upon dilution.

The hydrazone group, H_(z), must be converted to a chelator or bondingunit, C_(h), that is either a hydrazine group of the formula R⁴⁰ R⁴¹NNH₂ or a diazine group of formula R⁴⁰ N═NH, that may or may not beprotonated, in order for the chelator or bonding unit C_(h') to beformed with the metal radionuclide, M_(t). The chelator or bonding unit,C_(h), when bound to the metal radionuclide, M_(t), is designatedC_(h'). The conversion of the hydrazone group, H_(z), to the chelator orbonding unit, C_(h), can occur either prior to reaction with theradionuclide, in which case the radionuclide and the ancillary orco-ligand or ligands are combined not with the reagent but with ahydrolyzed form of the reagent bearing the chelator or bonding unit,C_(h), or in the presence of the radionuclide in which case the reagentitself is combined with the radionuclide and the ancillary or co-ligandor ligands. In the latter case, the pH of the reaction mixture must beneutral or acidic.

Radiopharmaceuticals of the formula (Q)_(d') L_(n) --C_(h') !_(x)--M_(t) (A_(L1))_(y) can be easily prepared from the reagents of thepresent invention by admixing a salt of a radionuclide, a reagent ofFormula 1, an ancillary ligand A_(L1), and a reducing agent, in anaqueous solution at temperatures from room temperature to 100° C.Alternatively, the radiopharmaceuticals can be prepared by firstadmixing a salt of a radionuclide, an ancillary ligand A_(L1), and areducing agent in an aqueous solution at temperatures from roomtemperature to 100° C. to form an intermediate radionuclide complex withthe ancillary ligand A_(L1) then adding a reagent of Formula 1 andreacting further at temperatures from room temperature to 100° C.

Radiopharmaceuticals of the formula (Q)_(d') L_(n) --C_(h') !_(x)--M_(t) (A_(L1))_(y) (A_(L2))_(z) can be easily prepared from thereagents of the present invention by admixing a salt of a radionuclide,a reagent of Formula 1, an ancillary ligand A_(L1), an ancillary ligandA_(L2), and optionally a reducing agent, in an aqueous solution attemperatures from room temperature to 100° C. Alternatively, theradiopharmaceuticals can be prepared by first admixing a salt of aradionuclide, an ancillary ligand A_(L1), a reagent of Formula 1, and areducing agent in an aqueous solution at temperatures from roomtemperature to 100° C. to form an intermediate radionuclide complex, andthen adding an ancillary ligand A_(L2) and reacting further attemperatures from room temperature to 100° C.

The total time of preparation will vary depending on the identity of theradionuclide, the identities and amounts of the reactants and theprocedure used for the preparation. The preparations may be complete,resulting in >80% yield of the radiopharmaceutical, in 1 minute or mayrequire more time. If higher purity radiopharmaceuticals are needed ordesired, the products can be purified by any of a number of techniqueswell known to those skilled in the art such as liquid chromatography,solid phase extraction, solvent extraction, dialysis or ultrafiltration.

The use of a reagent of the present invention for the synthesis of aradiopharmaceutical for imaging thrombosis, comprising a stablehydrazone-linker modified-cyclic IIb/IIIa receptor antagonist is shownin Scheme 5. The binary ligand system of the technetium-99m radionuclideis comprised of the diazenido bonding unit C_(h') and two tricineancillary ligands, A_(L1). The structure shown is only one of a numberof possible isomeric forms of the radiopharmaceutical due tocoordination isomerism of the diazenido bonding unit and the two tricineligands. ##STR15##

The use of a reagent of the present invention for the synthesis of aradiopharmaceutical for imaging thrombosis, comprising a stablehydrazone-linker modified-cyclic IIb/IIIa receptor antagonist, andhaving a ternary ligand system, is shown in Scheme 6. The ternary ligandsystem of the technetium-99m radionuclide is comprised of the diazenidobonding unit C_(h'), one tricine ancillary ligand, A_(L1), and onetrisubstituted phosphine ancillary ligand, A_(L2). The structure shownis one of two possible isomeric forms of the radiopharmaceutical due tocoordination isomerism of the diazenido bonding unit. ##STR16##

Radionuclides that can used with the reagents of the present inventionto synthesize radiopharmaceuticals are selected from the group ^(99m)Tc, ¹⁸⁶ Re, or ¹⁸⁸ Re. For diagnostic purposes ^(99m) Tc is thepreferred isotope. Its 6 hour half-life and 140 keV gamma ray emissionenergy are almost ideal for gamma scintigraphy using equipment andprocedures well established for those skilled in the art. The rheniumisotopes also have gamma ray emission energies that are compatible withgamma scintigraphy, however, they also emit high energy beta particlesthat are more damaging to living tissues. These beta particle emissionscan be utilized for therapeutic purposes, for example, cancerradiotherapy.

The technetium and rhenium radionuclides are preferably in the chemicalform of pertechnetate or perrhenate and a pharmaceutically acceptablecation. The pertechnetate salt form is preferably sodium pertechnetatesuch as obtained from commercial Tc-99m generators. The amount ofpertechnetate used to prepare the radiopharmaceuticals of the presentinvention can range from 0.1 mCi to 1 Ci, or more preferably from 1 to200 mCi.

The amount of the reagents of the present invention used to prepare theradiopharmaceuticals can range from 0.1 μg to 10 mg, or more preferablyfrom 0.5 μg to 100 μg. The amount used will be dictated by the amountsof the other reactants and the identity of the radiopharmaceuticals ofFormula 2 to be prepared.

The amounts of the ancillary ligands A_(L1) used can range from 0.1 mgto 1 g, or more preferrably from 1 mg to 100 mg. The exact amount for aparticular radiopharmaceutical is a function of identity of theradiopharmaceuticals of Formula 2 to be prepared, the procedure used andthe amounts and identities of the other reactants. Too large an amountof A_(L1) will result in the formation of by-products comprised oftechnetium labeled A_(L1) without a biologically active molecule orby-products comprised of technetium labeled biologically activemolecules with the ancillary ligand A_(L1) but without the ancillaryligand A_(L2). Too small an amount of A_(L1) will result in otherby-products such as reduced hydrolyzed technetium, or technetiumcolloid.

The amounts of the ancillary ligands A_(L2) used can range from 0.001 mgto 1 g, or more preferrably from 0.01 mg to 10 mg. The exact amount fora particular radiopharmaceutical is a function of the identity of theradiopharmaceuticals of Formula 2 to be prepared, procedure used and theamounts and identities of the other reactants. Too large an amount ofA_(L2) will result in the formation of by-products comprised oftechnetium labeled A_(L2) without a biologically active molecule orby-products comprised of technetium labeled biologically activemolecules with the ancillary ligand A_(L2) but without the ancillaryligand A_(L1).

A reducing agent can optionally be used for the synthesis of theradiopharmaceuticals of Formula 2 which are comprised of an ancillaryligand A_(L2). Suitable reducing agents include stannous salts,dithionite or bisulfite salts, borohydride salts, andformamidinesulfinic acid, wherein the salts are of any pharmaceuticallyacceptable form. The preferred reducing agent is a stannous salt. Theuse of a reducing agent is optional because the ancillary ligand A_(L2)can also serve to reduce the Tc-99m-pertechnetate. The amount of areducing agent used can range from 0.001 mg to 10 mg, or more preferablyfrom 0.005 mg to 1 mg.

Another aspect of the present invention are diagnostic kits for thepreparation of radiopharmaceuticals useful as imaging agents for thediagnosis of cardiovascular disorders, infectious disease, inflammatorydisease and cancer. Diagnostic kits of the present invention compriseone or more vials containing the sterile, non-pyrogenic, formulationcomprised of a predetermined amount of the reagent of formula (Q)_(d')--L_(n) --H_(z), one or two ancillary or co-ligands and optionally othercomponents such as reducing agents, transfer ligands, buffers,lyophilization aids, stabilization aids, solubilization aids andbacteriostats. The inclusion of one or more optional components in theformulation will frequently improve the ease of synthesis of theradiopharmaceutical by the practising end user, the ease ofmanufacturing the kit, the shelf-life of the kit, or the stability andshelf-life of the radiopharmaceutical. The improvement achieved by theinclusion of an optional component in the formulation must be weighedagainst the added complexity of the formulation and added cost tomanufacture the kit. The one or more vials that contain all or part ofthe formulation can independently be in the form of a sterile solutionor a lyophilized solid.

Buffers useful in the preparation of radiopharmaceuticals and indiagnostic kits useful for the preparation of said radiopharmaceuticalsinclude but are not limited to phosphate, citrate, sulfosalicylate, andacetate. A more complete list can be found in the United StatesPharmacopeia.

Lyophilization aids useful in the preparation diagnostic kits useful forthe preparation of radiopharmaceuticals include but are not limited tomannitol, lactose, sorbitol, dextran, Ficoll, andpolyvinylpyrrolidine(PVP).

Stabilization aids useful in the preparation of radiopharmaceuticals andin diagnostic kits useful for the preparation of saidradiopharmaceuticals include but are not limited to ascorbic acid,cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite,gentisic acid, and inositol.

Solubilization aids useful in the preparation of radiopharmaceuticalsand in diagnostic kits useful for the preparation of saidradiopharmaceuticals include but are not limited to ethanol, glycerin,polyethylene glycol, propylene glycol, polysorbates and lecithin.

Bacteriostats useful in the preparation of radiopharmaceuticals and indiagnostic kits useful for the preparation of said radiopharmaceuticalsinclude but are not limited to benzyl alcohol, benzalkonium chloride,chlorbutanol, and methyl, propyl or butyl paraben.

A component in a diagnostic kit can also serve more than one function. Areducing agent can also serve as a stabilization aid, a buffer can alsoserve as a transfer ligand, a lyophilization aid can also serve as atransfer, ancillary or co-ligand and so forth.

The predetermined amounts of each component in the formulation aredetermined by a variety of considerations that are in some casesspecific for that component and in other cases dependent on the amountof another component or the presence and amount of an optionalcomponent. In general, the minimal amount of each component is used thatwill give the desired effect of the formulation. The desired effect ofthe formulation is that the practising end user can synthesize theradiopharmaceutical and have a high degree of certainty that theradiopharmaceutical can be safely injected into a patient and willprovide diagnostic information about the disease state of that patient.

The diagnostic kits of the present invention will also contain writteninstructions for the practising end user to follow to synthesize theradiopharmaceuticals. These instructions may be affixed to one or moreof the vials or to the container in which the vial or vials are packagedfor shipping or may be a separate insert, termed the package insert.

Another aspect of the present invention contemplates a method of imagingthe site of thrombotic disease in a patient involving: (1) synthesizinga radiopharmaceutical, using a reagent of the present invention, thatlocalizes at the site of the thrombotic disease due to an interactionbetween the biologically active group, Q, of the radiopharmaceutical anda receptor or binding site expressed at the site of the disease or witha receptor or binding site on an endogenous blood component thataccumulates at the site; (2) administering said radiopharmaceutical to apatient by injection or infusion; (3) imaging the patient using eitherplanar or SPECT gamma scintigraphy.

Another aspect of the present invention contemplates a method of imagingthe site of infection or infectious disease in a patient involving: (1)synthesizing a radiopharmaceutical, using a reagent of the presentinvention, that localizes at the site of the infection or infectiousdisease due to an interaction between the biologically active group, Q,of the radiopharmaceutical and a receptor or binding site expressed atthe site of the disease or with a receptor or binding site on anendogenous blood component that accumulates at the site; (2)administering said radiopharmaceutical to a patient by injection orinfusion; (3) imaging the patient using either planar or SPECT gammascintigraphy.

Another aspect of the present invention contemplates a method of imagingthe site of inflammation in a patient involving: (1) synthesizing aradiopharmaceutical, using a reagent of the present invention, thatlocalizes at the site of the inflammation due to an interaction betweenthe biologically active group, Q, of the radiopharmaceutical and areceptor or binding site expressed at the site of the inflammation orwith a receptor or binding site on an endogenous blood component thataccumulates at the site; (2) administering said radiopharmaceutical to apatient by injection or infusion; (3) imaging the patient using eitherplanar or SPECT gamma scintigraphy.

Another aspect of the present invention contemplates a method of imagingthe site of cancer in a patient involving: (1) synthesizing aradiopharmaceutical, using a reagent of the present invention, thatlocalizes at the site of the cancer due to an interaction between thebiologically active group, Q, of the radiopharmaceutical and a receptoror binding site expressed at the site of the cancer or with a receptoror binding site on an endogenous blood component that accumulates at thesite; (2) administering said radiopharmaceutical to a patient byinjection or infusion; (3) imaging the patient using either planar orSPECT gamma scintigraphy.

EXAMPLE SECTION

The materials used to synthesize the reagents of the present inventiondescribed in the following examples were obtained as follows.Cyclo-(D-Val-NMeArg-Gly-Asp-Mamb(5-Aca)) andcyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hynic-5-Aca)) was synthesized asdescribed in co-pending U.S. Ser. No. 08/415,908,861 (equivalent to WO94/22494). The following were obtained from commercial sources and usedas received: hydrazinonicotinic acid, N-hydroxysuccinimide (NHS),dicyclohexylcarbodiimide (DCC), tricine,tris(3-sulfonatophenyl)phosphine trisodium salt (TPPTS), stannouschloride dihydrate, dimethylformamide (DMF), trifluoroacetic acid (TFA),acetonitrile, 4-pyridinecarboxaldehyde, ammonium acetate, sodiumdihydrogen phosphate, sodium 2-formylbenzenesulfonate, triethylamine,mannitol, crotonaldehye, 4-carboxybenzaldehyde, and glyoxylic acid.Deionized water was obtained from a Milli-Q Water System and was of >18MΩ quality. Tc-99m-pertechnetate (^(99m) TcO₄ ⁻) was obtained from aDuPont Pharma ⁹⁹ Mo/^(99m) Tc generator.

Example 1

Synthesis of the Benzaldehyde Hydrazone ofCyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)).

To a solution of 20 mg (0.0215 mmol) ofcyclo(D-Val-NMeArg-Gly-Asp-Mamb(5-Aca)).2TFA and 7.5 mg (0.0222 mmol) ofsuccinimidyl 6-(2-benzaldehydehydrazino)nicotinate in DMF (1 ml) wasadded Et₃ N (10 μl), and the reaction mixture was allowed to stir at RTfor 42 hours. The reaction mixture was concentrated, dissolved in 50%CH₃ CN/H₂ O, and lyophilized to provide the crude title compound (23.5mg) as an off-white powder. Purification was accomplished byreversed-phase HPLC on a preparative Vydac C18 column (2.5×25 cm) usinga gradient of 6 to 72% acetonitrile containing 0.1% trifluoroacetic acidat a flow rate of 15 ml/min to give the TFA salt of the title compound(17.5 mg, 71%) as a fluffy white solid; ¹ H NMR (D₆ -DMSO) 11.30 (br s,OH), 10.02 (s, NH), 8.94 (d, 1H), 8.61 (d, 1H), 8.55 (d, 1H), 8.41 (m,2H), 8.10 (s, ═CH), 8.09 (m, 1H), 7.70 (m, 4H), 7.61 (m, 1H), 7.52 (t,1H), 7.42 (m, 3H), 7.27 (d, 1H), 7.07 (s, 1H), 5.18 (dd, 1H), 4.53 (m,2H), 4.34 (dd, 1H), 4.20 (dd, 1H), 4.02 (dd, 1H), 3.25 (q, 2H), 3.13 (q,2H), 2.99 (s, NCH₃), 2.72 (dd, 1H), 2.50 (m, 1H), 2.33 (t, 2H), 2.10 (m,2H), 1.60 (m, 5H), 1.35 (m, 4H), 1.10 (d, CH₃), 0.92 (d, CH₃);FAB(NBA)-MS: M+H!=926.4625 (Calcd for C₄₅ H₆₀ N₁₃ O₉ =926.4637).

Example 2

Synthesis of the 2-Formylbenzenesulfonic Acid Hydrazone ofCyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)).

Sodium 2-formylbenzenesulfonate (3.9 mg, 0.019 mmol) andcyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)) (10 mg,0.0094 mmol) were dissolved in 0.05M sodium phosphate buffer, pH 7.0(1.0 mL) and allowed to stand at ambient temperature for 1.5 h, at whichtime the entire reaction had turned to a gel. The gel was dissolved in1.0 mL of 10% acetonitrile containing 0.1M NH₄ OAc and purified usingreversed-phase HPLC with a preparative Zorbax-RX C18 column (21.2×250mm) at a flow rate of 15 mL/min using 10% acetonitrile containing 0.1MNH₄ OAc for two minutes, followed by a 4.44%/min gradient of 10 to 50%acetonitrile containing 1.1M NH₄ OAc. The product fraction waslyophilized to give the title compound as a fluffy colorless solid (7mg, 74%). Analytical HPLC with a Zorbax-RX C18 column (4.6×250 mm) at aflow rate of 1.5 mL/min using a 4.0%/min gradient of 10 to 50%acetonitrile containing 0.05M NH₄ OAc indicated a product purity of97.3%. DCI-MS: M+H!=1006.3.

Example 3

Synthesis of the p-Dimethylaminobenzaldehyde Hydrazone ofCyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)).

The title compound was prepared by the general procedure described abovefor the benzaldehyde hydrazone ofcyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)) (Example 1).Coupling of the cyclic compound (32 mg, 0.0344 mmol) and succinimidyl6-(2-(4-dimethylamino)benzaldehydehydrazino)nicotinate (13.5 mg, 0.0354mmol) provided the crude title compound (47 mg) as a yellow powder.Purification was accomplished by reversed-phase HPLC on a preparativeVydac C18 column (2.5×25 cm) using a gradient of 9 to 72% acetonitrilecontaining 0.1% trifluoroacetic acid at a flow rate of 15 ml/min to givethe TFA salt of the title compound (29.7 mg, 72%) as a fluffy whitesolid; ¹ H NMR (D₆ -DMSO) 10.03 (s, NH), 8.94 (d, 1H), 8.55 (d, 1H),8.50 (s, 1H), 8.42 (t, 1H), 8.15 (br s, 1H), 8.06 (s, 1H), 7.70 (d, 2H),7.61 (m, 4H), 7.16 (d, 1H), 7.07 (s, 1H), 7.00 (br s, 2H), 6.76 (d, 2H),5.17 (dd, 1H), 4.52 (m, 2H), 4.33 (dd, 1H), 4.20 (dd, 1H), 3.25 (q, 2H),3.12 (q, 2H), 2.98 (s, 3 NCH₃), 2.72 (dd, 1H), 2.50 (m, 1H), 2.33 (t,2H), 2.10 (m, 2H), 1.60 (m, 5H), 1.35 (m, 4H), 1.10 (d, CH₃), 0.92 (d,CH₃); FAB(NBA)-MS: M+H!=969.5043 (Calcd for C₄₇ H₆₅ N₁₄ O₉ =969.5059).

Example 4

Synthesis of the 4-Carboxybenzaldehyde Hydrazone ofCyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)).

A mixture ofcyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)). 2 HBr (50mg, 50 μmole) and 4-carboxybenzaldehyde (70.35 μmole) was stirred indimethylformamide (1 mL) at room temperature, under nitrogen, for 4hours. The solvent was removed under vacuum, the residue dissolved in amixture of acetonitrile-water, and lyophilized to dryness. The crudemixture was purified by reversed-phase HPLC with a preparative Zorbax-RXC18 column (21.2×250 mm) at a flow rate of 15 mL/min, using a mobilephase of solvent A (50 mM ammonium acetate), solvent B (50 mM ammoniumacetate in 50% acetonitrile) and the following gradient: The followinggradient was used: 0-2 min, 20% B; 30 min, 50% B (held until 32 min); 35min, 100% B(held until 38 min); 40 min, 20% B. Yield of purified product7 mg (14%). DCI-MS (High Resolution) M+H!=970.453526 (calculatedmolecular weight 969.445702.

Example 5

Synthesis of the Crotonaldehyde Hydrazone ofCyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca))

The title compound was synthesized as described for the4-carboxybenzaldehyde hydrazone ofcyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)) (Example 4)substituting crotonaldehyde for 4-carboxybenzaldehyde. The crudematerial was purified by preparative HPLC using the following gradient:0-2 min, 20% B; 4 min, 55% B (held until 5 min); 30 min, 70% B(helduntil 32 min); 35 min, 100% B (held until 38 min); 40 min, 20% B; togive 4.5 mg (10%) of the purified product. DCI-MS (High Resolution)M+H!=890.463696 (calculated molecular weight 889.455872.

Example 6

Synthesis of the Glyoxylic Acid Hydrazone ofCyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)).

The title compound was synthesized as described for the4-carboxybenzaldehyde hydrazone ofcyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)) (Example 4)substituting glyoxylic acid for 4-carboxybenzaldehyde. The crudematerial was purified by preparative HPLC using the following gradient:0-5 min, 20% B; 40 min, 50% B (held until 42 min); 45 min, 100% B(helduntil 46 min); 48 min, 20% B to give 4.6 mg (10%) of the purifiedproduct. DCI-MS (High Resolution) M+H!=894.422225 (calculated molecularweight 893.414402.

Example 7

Synthesis of the Acetophenone Hydrazone ofCyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)).

To a solution of 82 mg (0.1075 mmol) of crudecyclo-(D-Val-NMeArg-Gly-Asp-Mamb(5-Aca)).HOAc, TFA (16.6 μl, 0.215mmol), and 38 mg (0.1075 mmol) of succinimidyl6-(2-acetophenonehydrazino)nicotinate in DMF (5 ml) was added Et₃ N (75μl), and the reaction mixture was allowed to stir at RT for 42 hours.The reaction mixture was concentrated, dissolved in 50% CH₃ CN/H₂ O, andlyophilized to provide the crude title compound (130 mg) as a paleyellow powder. Purification of a portion of this material wasaccomplished by reversed-phase HPLC on a preparative Vydac C18 column(2.5×25 cm) using a gradient of 2 to 90% acetonitrile containing 0.1%trifluoroacetic acid at a flow rate of 15 ml/min to give the TFA salt ofthe title compound as a fluffy white solid; ¹ H NMR (D₆ -DMSO) 10.03 (s,NH), 8.93 (d, 1H), 8.62 (s, 1H), 8.55 (d, 1H), 8.42 (m, 2H), 8.13 (br s,1H), 7.87 (d, 2H), 7.70 (m, 2H), 7.55 (m, 2H), 7.40 (m, 4H), 7.07 (s,1H), 5.17 (dd, 1H), 4.52 (m, 2H), 4.33 (dd, 1H), 4.20 (dd, 1H), 4.02(dd, 1H), 3.63 (dd, 1H), 3.26 (q, 2H), 3.12 (q, 2H), 2.98 (s, NCH₃),2.72 (dd, 1H), 2.50 (m, 1H), 2.35 (s, CH₃), 2.33 (m, 2H), 2.10 (m, 2H),1.60 (m, 5H), 1.35 (m, 4H), 1.10 (d, CH₃), 0.92 (d, CH₃); FAB(NBA)-MS:M+H!=940.4818 (Calcd for C4₆ H₆₂ N₁₃ O₉ =940.4793).

Example 8

Synthesis of the 1-(Methoxycarbonyl)acetaldehyde Hydrazone ofCyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)).

The title compound was prepared by the general procedure described abovefor the acetophenonecyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)) hydrazone(Example 7). Coupling of the crude cyclic compound (82 mg, 0.1075 mmol)and succinimidyl6-(2-(1-methoxycarbonyl)acetaldehydehydrazino)nicotinate (36 mg, 0.1077mmol) provided the crude title compound (123 mg) as a pale yellowpowder. Purification of a portion of this material was accomplished byreversed-phase HPLC using the conditions described in Example 8 to givethe TFA salt of the title compound as a fluffy white solid; ¹ H NMR (D₆-DMSO) 10.69 (s, NH), 10.02 (s, NH), 8.92 (d, 1H), 8.70 (d, 1H), 8.55(d, 1H), 8.44 (m, 2H), 8.14 (dd, 1H), 7.70 (s, 2H), 7.56 (m, 2H), 7.28(d, 1H), 7.07 (s, 1H), 5.17 (dd, 1H), 4.52 (m, 2H), 4.33 (dd, 1H), 4.19(dd, 1H), 4.04 (m, 1H), 3.76 (s, OCH₃), 3.63 (dd, 1H), 3.26 (q, 2H),3.13 (q, 2H), 2.99 (s, NCH₃), 2.72 (dd, 1H), 2.50 (m, 1H), 2.33 (t, 2H),2.13 (s, CH₃), 1.60 (m, 5H), 1.35 (m, 4H), 1.10 (d, CH₃), 0.92 (d, CH₃);FAB-MS: M+H!=922.4539 (Calcd for C₄₂ H₆₀ N₁₃ O₁₁ =922.4535).

Example 9

Synthesis of the Cyclopentanone Hydrazone ofCyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)).

The title compound was prepared by the general procedure described abovefor the acetophenonecyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)) hydrazone(Example 7). Coupling of the crude cyclic compound (82 mg, 0.1075 mmol)and succinimidyl 6-(2-cyclopentanonehydrazino)nicotinate (35 mg, 0.1106mmol) provided the crude title compound (131 mg) as a pale yellowpowder. Purification of a portion of this material was accomplished byreversed-phase HPLC using the conditions described in Example 8 to givethe TFA salt of the title compound as a fluffy white solid; ¹ H NMR (D₆-DMSO) 10.02 (s, NH), 8.93 (d, 1H), 8.61 (d, 1H), 8.55 (d, 1H), 8.51 (s,1H), 8.41 (t, 1H), 8.10 (m, 1H), 7.70 (s, 2H), 7.55 (m, 1H), 7.52 (t,1H), 7.10 (br s, 3H), 7.06 (s, 1H), 5.17 (dd, 1H), 4.52 (m, 2H), 4.33(dd, 1H), 4.19 (dd, 1H), 4.02 (dd, 1H), 3.62 (d, 1H), 3.24 (q, 2H), 3.12(q, 2H), 2.99 (s, NCH₃), 2.72 (dd, 1H), 2.50 (m, 1H), 2.41 (m, 4H), 2.33(t, 2H), 2.10 (m, 2H), 1.75 (m, 3H), 1.68 (m, 4H), 1.34 (m, 4H), 1.10(d, CH₃), 0.92 (d, CH₃); FAB(NBA/TFA)-MS: M+H!=904.5136 (Calcd for C₄₃H₆₂ N₁₃ O₉ =904.4793).

Example 10

Synthesis of the 2-(Methoxycarbonyl)cyclopentanone Hydrazone ofCyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hynic-5-Aca))

The title compound was prepared by the general procedure described abovefor the acetophenonecyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)) hydrazone(Example 7). Coupling of the crude cyclic compound (82 mg, 0.1075 mmol)and succinimidyl6-(2-(2-methoxycarbonyl)cyclopentanonehydrazino)nicotinate (41 mg,0.1095 mmol) provided the crude title compound (138 mg) as a pale yellowpowder. Purification of a portion of this material was accomplished byreversed-phase HPLC using the conditions described in Example 8 to givethe TFA salt of the title compound as a fluffy white solid; ¹ H NMR (D₆-DMSO) 10.01 (s, NH), 8.90 (m, 1H), 8.57 (m, 2H), 8.39 (m, 2H), 8.07 (d,1H), 7.71 (s, 2H), 7.59 (m, 2H), 7.09 (m, 2H), 5.17 (dd, 1H), 4.52 (m,2H), 4.34 (dd, 1H), 4.20 (dd, 1H), 4.02 (d, 1H), 3.67 (S, OCH₃), 3.24(q, 2H), 3.12 (m, 2H), 2.99 (s, NCH₃), 2.71 (dd, 1H), 2.50 (m, 1H), 2.34(t, 2H), 2.10 (m, 4H), 1.60 (m, 5H), 1.34 (m, 3H), 1.25 (m, 2H), 1.10(d, CH₃), 0.93 (d, CH₃); ESI-MS: M+H!=962 (Calcd for C₄₅ H₆₄ N₁₃ O₁₁=962.4848).

Example 11

Synthesis of the 4-Pyridinecarboxaldehyde Hydrazone ofCyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hydrazinonicotinyl-5-Aca)).

4-Pyridinecarboxaldehyde (1.14 mg, 0.0106 mmol) andcyclo-(D-Val-NMeArg-Gly-Asp-Mamb(Hynic-5-Aca)) (10 mg, 0.0094 mmol) weredissolved in 0.05M sodium phosphate buffer, pH 7.0 (5.0 mL) and allowedto stand at ambient temperature for 72 h. The now slightly yellowsolution was lyophilized to dryness and the resulting solid was purifiedusing reversed-phase HPLC with a preparative Zorbax-RX C18 column(21.2×250 mm) at a flow rate of 15 mL/min using 10% acetonitrilecontaining 0.1M NH₄ OAc for two minutes, followed by a 4.44%/mingradient of 10 to 50% acetonitrile containing 0.1M NH₄ OAc. The productfraction (retention time 10-12 min) was lyophilized to give the titlecompound as a fluffy colorless solid (8 mg, 74%). Analytical HPLC with aZorbax-RX C18 column (4.6×250 mm) at a flow rate of 1.5 mL/min using a4.0%/min gradient of 10 to 50% acetonitrile containing 0.05M NH₄ OAcindicated a product purity of 98.7%.

The following examples illustrate the synthesis of stable hydrazonesbearing a chemically reactive moiety useful in the synthesis of thereagents described above.

Example 12

Synthesis of Succinimidyl 6-(2-Benzaldehydehydrazino)nicotinate

To a suspension of 6-hydrazinonicotinic acid (1.00 g, 6.5 mmol) in DMF(40 ml) was added benzaldehyde (0.70 ml, 6.9 mmol), and the reactionmixture was allowed to stir at room temperature for 3 hours. To thehomogeneous reaction mixture was added N-hydroxysuccinimide (752 mg, 6.5mmol) and DCC (3.00 ml, 13.4 mmol), and the reaction mixture was allowedto stir at room temperature for 18 hours. The reaction mixture wasfiltered, concentrated, diluted with EtOAc (50 ml), and the mixture washeated at reflux for 1 hour. Filtration of the hot mixture provided thetitle compound (1.78 g, 81%) as a pale yellow powder. This material wasused without further purification. ¹ H NMR (D₆ -DMSO) 11.86 (s, NH),8.82 (dd, Py-H), 8.20 (dd, Py-H), 8.20 (s, ═CH), 7.75 (dd, 2 Ar-H), 7.43(m, Py-H & 3 Ar-H), 2.89 (s, 2 CH₂); DCI(NH₃)-MS: M+H!=339.1084 (Calcdfor C₁₇ H₁₅ N₄ O₄ =339.1093).

Example 13

Synthesis of Succinimidyl 6-(2-Acetophenonehydrazino)nicotinate

The title compound was prepared by the general procedure described abovefor succinimidyl 6-(2-benzaldehydehydrazino)nicotinate (Example 12).Filtration of the EtOAc mixture provided the title compound (1.26 g,55%, contains traces of DCU) as an off-white powder. A pure sample ofthe title compound (853 mg, 37%) was obtained from the filtrate asgolden crystals. ¹ H NMR (D₆ -DMSO) 10.86 (s, NH), 8.84 (dd, Py-H), 8.21(dd, Py-H), 7.86 (dd, 2 Ar-H), 7.41 (m, Py-H & 3 Ar-H), 2.89 (s, 2 CH₂),2.39 (s, CH₃); DCI(NH₃)-MS: M+H!=(Calcd for C₁₈ H₁₇ N₄ O₄ =353.1250).

Example 14

Synthesis of Succinimidyl6-(2-(4-Dimethylamino)benzaldehydehydrazino)nicotinate

The title compound was prepared by the general procedure described abovefor succinimidyl 6-(2-benzaldehydehydrazino)nicotinate (Example 12),except that only 1 equivalent of DCC (1.5 ml, 6.7 mmol) was used. Hotfiltration of the EtOAc mixture provided the title compound (1.20 g,48%) as a yellow powder. This material was used without furtherpurification. ¹ H NMR (D₆ -DMSO) 11.58 (s, NH), 8.76 (dd, Py-H), 8.13(dd, Py-H), 8.07 (s, ═CH), 7.54 (d, 2 Ar-H), 7.29 (d, Py-H), 6.75 (d, 2Ar-H), 2.97 (s, 2 NCH₃), 2.88 (s, 2 CH₂); DCI(NH₃)-MS: M+H!=382.1513(Calcd for C₁₉ H₂₀ N₅ O₄ =382.1515).

Example 15

Synthesis of Succinimidyl6-(2-(1-Methoxycarbonyl)acetaldehydehydrazino)nicotinate

The title compound was prepared by the general procedure described abovefor succinimidyl 6-(2-benzaldehydehydrazino)nicotinate (Example 12).Filtration of the EtOAc mixture provided the title compound (552 mg,25%, contains traces of DCU) as a pale white powder. This material wasused without further purification. Concentration of the filtrate, andtrituration with EtOAc provided the title compound (349 mg, 16%,contains traces of DCU). ¹ H NMR (D₆ -DMSO) 11.21 (s, NH), 8.91 (dd,Py-H), 8.33 (dd, Py-H), 7.42 (d, Py-H), 3.78 (s, OCH₃), 2.89 (s, 2 CH₂),2.18 (s, CH₃); DCI(NH₃)-MS: M+H!=(Calcd for C₁₄ H₁₅ N₄ O₆ =335.0991).

Example 16

Synthesis of Succinimidyl 6-(2-Cyclopentanonehydrazino)nicotinate

The title compound was prepared by the general procedure described abovefor succinimidyl 6-(2-benzaldehydehydrazino)nicotinate (Example 12).Filtration of the EtOAc mixture provided the title compound (1.78 g,86%, contains traces of DCU) as a pale yellow powder. Recrystallizationof this material from EtOAc provided a purified sample of the titlecompound (530 mg, contains traces of DCU). This material was usedwithout further purification. ¹ H NMR (D₆ -DMSO) 10.33 (s, NH), 8.76(dd, Py-H), 8.11 (dd, Py-H), 7.15 (d, Py-H), 2.88 (s, 2 CH₂), 2.41 (q, 2CH₂), 1.75 (m, 2 CH₂); DCI(NH₃)-MS: M+H!=(Calcd for C₁₅ H₁₇ N₄ O₄=317.1250).

Example 17

Synthesis of Succinimidyl6-(2-(2-Methoxycarbonyl)cyclopentanonehydrazino)nicotinate

The title compound was prepared by the general procedure described abovefor succinimidyl 6-(2-benzaldehydehydrazino)nicotinate (Example 12).Filtration of the EtOAc mixture provided the title compound (627 mg,26%, contains traces of DCU) as an off-white powder. Concentration ofthe filtrate, and trituration with EtOAc provided the title compound(1.17 g, 48%, contains traces of DCU). This material was used withoutfurther purification. ¹ H NMR (D₆ -DMSO) 10.56 (s, NH), 8.79 (dd, Py-H),8.15 (dd, Py-H), 7.11 (d, Py-H), 3.67 (s, OCH₃), 3.55 (t, CH), 2.88 (s,2 CH₂), 2.50 (m, CH₂), 1.90 (m, 2 CH₂); DCI(NH₃)-MS: M+H!=(Calcd for C₁₇H₁₉ N₄ O₆ =375.1304).

Example 18

Synthesis of Succinimidyl 6-(2-(2-Sulfo)benzaldehydehydrazino)nicotinateSodium Salt

The title compound was prepared by the general procedure described abovefor succinimidyl 6-(2-benzaldehydehydrazino)nicotinate (Example 12).Filtration of the EtOAc mixture provided a yellow solid, half of thismaterial was diluted with EtOAc (50 ml), and the mixture was heated atreflux for 1 hour. Filtration of the hot mixture provided the titlecompound (1.63 g, 85%) as a pale yellow powder. This material was usedwithout further purification. ¹ H NMR (D₆ -DMSO) 11.91 (s, NH), 9.16 (s,═CH), 8.79 (dd, Py-H), 8.16 (dd, Py-H), 8.03 (dd, Ar-H), 7.79 (dd,Ar-H), 7.35 (m, Py-H & 2 Ar-H), 2.88 (s, 2 CH₂); FAB(NBA)-MS:M+H!=419.2240 (Calcd for C₁₇ H₁₅ N₄ O₇ S=419.0661); Anal. Calcd for C₁₇H₁₄ NaN₄ O₇ S.(H₂ O)₁.5 : C, 43.69; H, 3.45; N, 11.99; Na, 4.92; S,6.86. Found: C, 43.62, 43.71; H, 3.59, 3.64; N, 12.13, 12.08; Na, 4.83,4.67; S, 6.56, 6.30.

Hydrazone Stability Testing

The stability of the reagents of the present invention were tested bycombining a solution of the reagents and a solution of formaldehyde andmonitoring the mixture by HPLC using Method 1.

    ______________________________________                                        HPLC Method 1:                                                                Column:              Zorbax Rx C18 (4.6 mm × 25 cm)                     Column Temp.:        50° C.                                            Flow Rate:           1.5 mL/min                                               Solvent A:           50 mM ammonium acetate                                   Solvent B:           50/50 50 mM ammonium                                                          acetatelacetonitrile                                     Gradient:  t = 0      20% B                                                              t = 20 min                                                                              100% B                                                              t = 22 min                                                                              100% B                                                              t = 23 min                                                                               20% B                                                   Wavelength:          240 nm                                                   ______________________________________                                    

The reagent of Example 1 was dissolved in 0.05M phosphate buffer, pH 7,(0.1 mg/mL) and 10 equivalents of formaldehyde (0.1 M in phosphatebuffer) added. The reaction mixture was analyzed every 0.5 h by HPLC.The change in peak area for the reagent of Example 1, expressed as apercentage of the initial value prior to formaldehyde addition, is shownin FIGURE 1. For comparison, the lower alkyl hydrazone,cyclo-(D-Val-NMeArg-Gly-Asp-Mamb(hydrazino-nicotinyl-5-Aca))propionaldehyde hydrazone was also tested. The reagent of Example 1 doesnot react with formaldehyde over 2.5 h whereas >90% of the lower alkylhydrazone reacts over 2 h.

In addition, a number of the reagents were generated in situ by reactionof 1 equivalent of the respective aldehyde or ketone withCyclo(D-Val-NMeArg-Gly-Asp-Mamb(hydrazinonicotinyl-5-Aca)) in 0.05Mphosphate buffer, pH 7.0 (0.1 mg/mL) and then their stability tested byaddition of one equivalent of formaldehyde and monitoring the solutionby HPLC. The stability test results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Formaldehyde Stability of Reagents                                                           Formaldehyde Test                                              Example        % Decrease*                                                    ______________________________________                                         1             0                                                               2             0                                                               3             0                                                               4             0                                                               5             0                                                               6             <1                                                              9             0                                                              11             0                                                              phenylacetaldehyde                                                                           25                                                             glycolaldehyde 40                                                             Lower Alkyl    77                                                             ______________________________________                                         *at 2 h                                                                  

The amounts of the reagents of Examples 1-6, 9 and 11 decrease <1% over2 h of exposure to one equivalent of formaldehyde indicating that theyare very stable. In contrast, the amount of the lower alkyl hydrazone,cyclo-(D-Val-NMeArg-Gly-Asp-Mamb(hydrazino-nicotinyl-5-Aca))propionaldehyde hydrazone, designated Lower Alkyl, decreases by 77%under the same conditions. Also included in Table 1 are the results fortwo other hydrazones,cyclo-(D-Val-NMeArg-Gly-Asp-Mamb(hydrazinonicotinyl-5-Aca))phenylacetaldehyde hydrazone, designated phenylacetaldehyde, andcyclo-(D-Val-NMeArg-Gly-Asp-Mamb(hydrazinonicotinyl-5-Aca))glycolaldehyde hydrazone, designated glycolaldehyde, the structures ofwhich are not circumscribed by this invention. The phenylacetaldehydehydrazone, in which there is a benzyl group attached to the methylenecarbon atom of the hydrazone, shows somewhat better stability,decreasing by 25%, while the glycolaldehyde hydrazone, in which there isa hydroxymethyl group attached to the methylene carbon atom of thehydrazone, shows marginally improved stability, decreasing by 40%. Thesedata show that for a hydrazone to be very stable, as are the reagents ofthe present invention, there must either be a conjugated π-system or thehydrazone must be part of a ring system. The phenyl group of thephenylacetaldehyde hydrazone is not conjugated to the C═N bond becauseit is one carbon atom removed. The glycolaldehyde hydrazone does notcontain another π-system.

The synthesis of a radiopharmaceutical of Formula 2 useful as a thrombusimaging agent from the reagents described in the preceeding examples canbe performed as described below.

Radiolabeling of Reagents Using Stannous Chloride

To a 10 mL vial was added 0.4 mL of tricine solution (40 mg) in H₂ O,followed by 0.2 mL of reagent solution in H₂ O (10-20 μg), 0.5 mL of^(99m) TcO₄ - solution (˜50 mCi), 0.2 mL of TPPTS solution (1 mg) in H₂O and 20 μl of SnCl₂.2H₂ O solution (20 μg in 0.1N HCl). The pH of thesolution was adjusted to 4 if necessary. The reaction mixture was heatedat 50°-80° C. for 30 min, and was analyzed by radio-HPLC using Method 2or 3.

Radiolabeling of Reagents without Stannous Chloride

To a 10 mL vial was added 0.1 mL tricine solution (10 mg) in H₂ O, 0.4mL reagent solution (20-40 μg) in H₂ O, 0.5 mL ^(99m) TcO₄ - (˜50 mCi)in saline, 0.2 mL mannitol solution (20 mg) in H₂ O, and 0.20 mL TPPTSsolution (7.0 mg) in H₂ O. The pH was adjusted to 4 using 0.1N HCl. Thereaction mixture was heated at 50°-80° C. for 30 min and then analyzedby radio-HPLC using Method 2 or 3.

    ______________________________________                                        HPLC Method 2                                                                 Column: Vydac C.sub.18, 250 mm × 4.6 mm, 300 Å pore                 size                                                                          Solvent A: 10 mM sodium monophosphate, pH 6.0                                 Solvent B: 100% acetonitrile                                                  Gradient:                                                                     0% B       30% B       75% B      0% B                                        0'         15'         25'        30'                                         Flow: 1.0 mL/min                                                              Detection by NaI probe                                                        HPLC Method 3                                                                 Column: Vydac C.sub.18, 250 mm × 4.6 mm, 300 Å pore                 size                                                                          Solvent A: 10 mM sodium monophosphate, pH 6.0                                 Solvent B: 100% acetonitrile                                                  Column Temperature 50° C.                                              Gradient:                                                                     5% B     13% B     20% B     75% B   5% B                                     0'       15'       20'       25'     30'                                      Flow: 1.0 mL/min                                                              Detection by NaI probe                                                        ______________________________________                                    

The radiopharmaceutical, ^(99m)Tc(tricine)(TPPTS)(cyclo(D-Val-NMeArg-Gly-Asp-Mamb(hydrazinonicotinyl-5-Aca)),formed is identical to that described in Example 1 of copending U.S.Ser. No. 08/415,908, synthesized from the non-hydrazone reagentCyclo(D-Val-NMeArg-Gly-Asp-Mamb(hydrazinonicotinyl-5-Aca), the synthesisof which is described in co-pending U.S. Ser. No. 08/415,908,861, thatwas demonstrated to have utility as a thrombus imaging agent. Theradiopharmaceutical is formed in >80% yield (based on Tc-99m) using thereagents described in Examples 1-6 and 8.

                  TABLE 2                                                         ______________________________________                                        Radiopharmaceutical Yields Using Reagents                                                           RCF                                                     Example               %                                                       ______________________________________                                        1                     94                                                      2                     91                                                      3                     86*                                                     4                     87                                                      5                     83                                                      6                     91*                                                     7                     45                                                      8                     87                                                      11                    30*                                                     ______________________________________                                         *heated at 50° C.                                                 

The fact that the radiopharmaceutical can be formed in good yield fromthe reagents of the present invention is a surprising result since thereagents have been demonstrated to be very stable and yet they must behydrolyzed in situ for the radiopharmaceutical to be formed. Thereagents of the present invention do not react with the aldehydes andketones that are frequently encountered in a pharmaceuticalmanufacturing setting, such as used in the manufacture of diagnostickits, and thus maintain their purity during the manufacturing process inmarked contrast to reagents comprised of lower alkyl hydrazones.

We claim:
 1. A reagent having the formula:

    (Q).sub.d' L.sub.n --H.sub.z,

and pharmaceutically acceptable salts thereof wherein, Q is a biologically active group selected from the group: peptides, polypeptides, and peptidomimetics; d' is 1-20; L_(n) is a linking group of formula:

    M.sup.1 -- Y.sup.1 (CR.sup.55 R.sup.56).sub.f" Y.sup.2 !.sub.f' --M.sup.2,

wherein: M¹ is -- (CH₂)_(g) Z¹ !_(g') --(CR⁵⁵ R⁵⁶)_(g") --; M² is --(CR⁵⁵ R⁵⁶)_(g") -- Z¹ (CH₂)_(g) !_(g') --; g is independently 0-10; g' is independently 0-1; g" is independently 0-10; f is independently 0-10; f' is independently 0-10; f" is independently 0-1; Y¹ and Y², are independently selected at each occurrence from: a bond, O, NR⁵⁶, C═O, C(═O)O, OC(═O)O, C(═O)NH--, C═NR⁵⁶, S, SO, SO₂, SO₃, NHC(═O), (NH)₂ C(═O), (NH)₂ C═S; Z¹ is independently selected at each occurrence from a C₆ -C₁₄ saturated, partially saturated, or aromatic carbocyclic ring system, substituted with 0-4 R⁵⁷ ; and a heterocyclic ring system, substituted with 0-4 R⁵⁷ ;R⁵⁵ and R⁵⁶ are independently selected at each occurrence from: hydrogen; C₁ -C₁₀ alkyl substituted with 0-5 R⁵⁷ ; alkaryl wherein the aryl is substituted with 0-5 R⁵⁷ ; R⁵⁷ is independently selected at each occurrence from the group: hydrogen, OH, NHR⁵⁸, C(═O)R⁵⁸, OC(═O)R⁵⁸, OC(═O)OR⁵⁸, C(═O)OR⁵⁸, C(═O)NR⁵⁸, C.tbd.N, SR⁵⁸, SOR⁵⁸, SO₂ R⁵⁸, NHC(═O)R⁵⁸, NHC(═O)NHR⁵⁸, NHC(═S)NHR⁵⁸ ; or, alternatively, when attached to an additional molecule Q, R⁵⁷ is independently selected at each occurrence from the group: O, NR⁵⁸, C═O, C(═O)O, OC(═O)O, C(═O)N--, C═NR⁵⁸, S, SO, SO₂, SO₃, NHC(═O), (NH)₂ C(═O), (NH)₂ C═S; and, R⁵⁸ is independently selected at each occurrence from the group: hydrogen; C₁ -C₆ alkyl; benzyl, and phenyl;H_(z) is a stable hydrazone of formula: ##STR17## wherein, R⁴⁰ is independently selected at each occurrence from the group: a bond to L_(n), C₁ -C₁₀ alkyl substituted with 0-3 R⁵², aryl substituted with 0-3 R⁵², cycloaklyl substituted with 0-3 R⁵², heterocycle substituted with 0-3 R⁵², heterocycloalkyl substituted with 0-3 R⁵², aralkyl substituted with 0-3 R⁵² and alkaryl substituted with 0-3 R⁵² ; R⁴¹ is independently selected from the group: hydrogen, aryl substituted with 0-3 R⁵², C₁ -C₁₀ alkyl substituted with 0-3 R⁵², and a heterocycle substituted with 0-3 R⁵² ;R⁵² is independently selected at each occurrence from the group: a bond to L_(n), ═O, F, Cl, Br, I,--CF₃,--CN, --CO₂ R⁵³, --C (═O)R⁵³, --C (═O)N (R⁵³)₂, --CHO, --CH₂ OR⁵³, --OC(═O)R⁵³, --OC(═O)OR^(53a), --OR⁵³, --OC(═O)N(R⁵³)₂, --NR⁵³ C (═O) R⁵³, --NR⁵⁴ C (═O)OR^(53a), --NR⁵³ C(═O)N(R⁵³)₂, --NR⁵⁴ SO₂ N(R⁵³)₂, --NR⁵⁴ SO₂ R^(53a), --SO₃ H, --SO₂ R^(53a), --SR⁵³, --S(═O)R^(53a), --SO₂ N(R⁵³)₂, --N(R⁵³)₂, --NHC(═NH)NHR⁵³, --C(═NH)NHR⁵³, ═NOR⁵³, NO₂, --C(═O)NHOR⁵³, --C (═O)NHNR⁵³ R^(53a), --OCH₂ CO₂ H, 2-(1-morpholino)ethoxy; R⁵³, R^(53a), and R⁵⁴ are each independently selected at each occurrence from the group: hydrogen, C₁ -C₆ alkyl, and a bond to L_(n) ; R⁸⁰ and R⁸¹ are independently selected from the group: H; C₁ -C₁₀ alkyl; --CN; --CO₂ R⁸⁵ ; --C(═O)R⁸⁵ ; --C(═O)N(R⁸⁵)₂ ; C₂ -C₁₀ 1-alkene substituted with 0-3 R⁸⁴ ; C₂ -C₁₀ 1-alkyne substituted with 0-3 R⁸⁴ ; aryl substituted with 0-3 R⁸⁴ ; unsaturated heterocycle substituted with 0-3 R⁸⁴ ; and unsaturated carbocycle substituted with 0-3 R⁸⁴ ; provided that when one of R⁸⁰ and R⁸¹ is H or alkyl, then the other is not H or alkyl; or, alternatively, R⁸⁰ and R⁸¹, may be taken together with the shown divalent carbon radical to form: ##STR18## wherein: R⁸² and R⁸³ may be independently selected from the group: H; R⁸⁴ ; C₁ -C₁₀ alkyl substituted with 0-3 R⁸⁴ ; C₂ -C₁₀ alkenyl substituted with 0-3 R⁸⁴ ; C₂ -C₁₀ alkynyl substituted with 0-3 R⁸⁴ ; aryl substituted with 0-3 R⁸⁴ ; heterocycle substituted with 0-3 R⁸⁴ ; and carbocycle substituted with 0-3 R⁸⁴ ;or, alternatively, R⁸², R⁸³ may be taken together to form a fused aromatic or heterocyclic ring; a and b indicate the positions of optional double bonds and n is 0 or 1,R⁸⁴ is independently selected at each occurrence from the group: ═O, F, Cl, Br, I, --CF₃, --CN, --CO₂ R⁸⁵, --C(═O)R⁸⁵, --C(═O)N(R⁸⁵)₂, --N(R⁸⁵)₃ ⁺ --CH₂ OR⁸⁵, --OC (═O)R⁸⁵, --OC(═O)OR^(85a), --OR⁸⁵, --OC(═O)N(R⁸⁵)₂, --NR⁸⁵ C(═O)R⁸⁵, --NR⁸⁶ C(═O)OR^(85a), --NR⁸⁵ C(═O)N(R⁸⁵)₂, --NR⁸⁶ SO₂ N(R⁸⁵)₂, --NR⁸⁶ SO₂ R^(85a), --SO₃ H, SO₃ Na, --SO₂ R^(85a), --SR⁸⁵, --S(═O)R^(85a), --SO₂ N(R⁸⁵)₂, --N(R⁸⁵)₂, --NHC(═NH)NHR⁸⁵, --C(═NH)NHR⁸⁵, ═NOR⁸⁵, --C(═O)NHOR⁸⁵, --OCH₂ CO₂ H, 2-(1-morpholino)ethoxy; and R⁸⁵, R^(85a), and R⁸⁶ are each independently selected at each occurrence from the group: hydrogen, C₁ -C₆ alkyl.
 2. The reagent of claim 1 wherein:Q is a biologically active molecule selected from the group: IIb/IIIa receptor antagonists, IIb/IIIa receptor ligands, fibrin binding peptides, leukocyte binding peptides, chemotactic peptides, somatostatin analogs, and selectin binding peptides; d' is 1 to 3; L_(n) is:--(CR⁵⁵ R⁵⁶)_(g") -- Y¹ (CR⁵⁵ R⁵⁶)_(f) Y² !_(f') --(CR⁵⁵ R⁵⁶)_(g") --,wherein: g" is 0-5; f is 0-5; f' is 1-5; Y¹ and Y², are independently selected at each occurrence from: O, NR⁵⁶, C═O, C(═O)O, OC(═O)O, C(═O)NH--, C═NR⁵⁶, S, SO, SO₂, SO₃, NHC(═O), (NH)₂ C(═O), (NH)₂ C═S; R⁵⁵ and R⁵⁶ are independently selected at each occurrence from: hydrogen, C₁ -C₁₀ alkyl, and alkaryl; H_(z) is a stable hydrazone of formula: ##STR19## wherein, R⁴⁰ is independently selected at each occurrence from the group: aryl substituted with 0-3 R⁵², and heterocycle substituted with 0-3 R⁵² ; R⁴¹ is independently selected from the group: hydrogen, aryl substituted with 0-1 R⁵², C₁ -C₃ alkyl substituted with 0-1 R⁵², and a heterocycle substituted with 0-1 R⁵² ;R⁵² is independently selected at each occurrence from the group: a bond to L_(n), --CO₂ R⁵³, --CH₂ OR⁵³, --SO₃ H, --SO₂ R^(53a), --N(R⁵³)₂, --NHC(═NH)NHR⁵³, and --OCH₂ CO₂ H; R⁵³, R^(53a) are each independently selected at each occurrence from the group: hydrogen and C₁ -C₃ alkyl; R⁸⁰ is independently selected at each occurrence from the group: --CO₂ R⁸⁵ ; C₂ -C₅ 1-alkene substituted with 0-3 R⁸⁴ ; C₂ -C₅ 1-alkyne substituted with 0-3 R⁸⁴ ; aryl substituted with 0-3 R⁸⁴ ; unsaturated heterocycle substituted with 0-3 R⁸⁴ ; R⁸¹ is independently selected at each occurrence from the group: H and C₁ -C₅ alkyl; or, alternatively, R⁸⁰ and R⁸¹, when taken together with the indicated divalent carbon radical form ##STR20## wherein R⁸² and R⁸³ may be independently selected from the group: H and R⁸⁴ ;or, alternatively, R⁸², R⁸³ may be taken together to form a fused aromatic or heterocyclic ring; a and b indicate the positions of optional double bonds and n is 0 or 1,R⁸⁴ is independently selected at each occurrence from the group: --CO₂ R⁸⁵, --C(═O)N(R⁸⁵)₂, --CH₂ OR⁸⁵, --OC(═O)R⁸⁵ --OR⁸⁵, --SO₃ H, --SO₃ Na, --N(R⁸⁵)₂, --OCH₂ CO₂ H; R⁸⁵ is independently selected at each occurrence from the group: hydrogen, C₁ -C₃ alkyl.
 3. The reagent of claim 2 wherein:Q represents a biologically active molecule selected from the group: IIb/IIIa receptor antagonists and chemotactic peptides;d' is 1; L_(n) is: --(CR⁵⁵ R⁵⁶)_(g") -- Y¹ (CR⁵⁵ R⁵⁶)_(f) Y² !_(f') --(CR⁵⁵ R⁵⁶)_(g") --,wherein: g" is 0-5; f is 0-5; f' is 1-5; Y¹ and Y², at each occurrence, are independently selected from: O, NR⁵⁶, C═O, C(═O)O, OC(═O)O, C(═O)NH--, C═NR⁵⁶, S, NHC(═O), (NH)₂ C(═O), (NH)₂ C═S; R⁵⁵ and R⁵⁶ are hydrogen; H_(z) is a stable hydrazone of formula: ##STR21## wherein, R⁴⁰ is independently selected at each occurrence from the group: heterocycle substituted with R⁵² ;R⁴¹ is hydrogen; R⁵² is a bond to L_(n) ; R⁸⁰ is independently selected from the group: --CO₂ R⁸⁵ ; C₂ -C₃ 1-alkene substituted with 0-1 R⁸⁴ ; aryl substituted with 0-1 R⁸⁴ ; unsaturated heterocycle substituted with 0-1 R⁸⁴ ; R⁸¹ is H; R⁸⁴ is independently selected at each occurrence from the group: --CO₂ R⁸⁵ ; --OR⁸⁵ ; --SO₃ H; SO₃ Na; --N(R⁸⁵)₂ ; R⁸⁵ is independently selected at each occurrence from the group:H and methyl.
 4. A reagent according to claim 3, wherein Q is a cyclic IIb/IIIa receptor antagonist.
 5. The reagents of claim 4 that are ##STR22##
 6. A kit for preparing a radiopharmaceutical comprising:(a) a predetermined quantity of a sterile, pharmaceutically acceptable reagent of any of claims 1-3 and 4, (b) a predetermined quantity of one or more sterile, pharmaceutically acceptable ancillary ligand(s); (c) a predetermined quantity of a sterile, pharmaceutically acceptable reducing agent; and (d) optionally, a predetermined quantity of a sterile, pharmaceutically acceptable component selected from the group:transfer ligands, buffers, lyophilization aids, stabilization aids, solubilization aids and bacteriostats.
 7. A kit for preparing a radiopharmaceutical comprising:(a) a predetermined quantity of a sterile, pharmaceutically acceptable reagent of any of claims 1-3 and 4, (b) a predetermined quantity of two sterile, pharmaceutically acceptable ancillary ligand(s); (c) a predetermined quantity of a sterile, pharmaceutically acceptable reducing agent; and (d) optionally, a predetermined quantity of a sterile, pharmaceutically acceptable component selected from the group: transfer ligands, buffers, lyophilization aids, stabilization aids, solubilization aids and bacteriostats. 